bluuebunny/crossref_metadata_2025 huggingface dataset:
CCR10 regulates maintenance and function of resident regulatory and effector T cells in the skin (MUC7P.772)
Authors: FU Yaoyao , Xia Mingcan , Hu Shaomin , McDowell Mary , Sarkar Surojit , Kalia Vandana , Xiong Na
Published: January 2023

Skin, the largest organ of human body, is under frequent assaults by various environmental agents. To maintain its integrity and function, immune cells in the skin are tightly regulated to allow tolerance to harmless antigens but mount effective response to dangerous assaults, and their dysregulation might increase incidence of skin diseases. CCR10, with its ligand CCL27 uniquely expressed by keratinocytes, are the most skin-specific chemokine/receptor pair implicated in the skin T cell migration and inflammatory diseases. However, targeting CCR10 or CCL27 had little effects on these processes in animal models and the regulation and function of CCR10 in vivo remain elusive. Using CCR10 knockout/EGFP knockin mice we found that CCR10-/- mice had imbalanced numbers and dysregulated functions of skin-resident effector and regulatory T (Teff and Treg) cells associated with enhanced innate and memory skin immune responses to antigen stimulations. On the other hand, CCR10 -/- mice cleared the skin Leishmania major (L. major) infection more efficiently than wild type (WT) mice did. Adoptive transfer studies found that CCR10 is required for the migration and maintenance of skin-resident CD8+ and CD4+ cells into homeostatic skin but not during the inflammation. These findings reveal that the CCR10/CCL27 axis plays important roles in the migration, maintenance and function of skin-resident regulatory and effector T cells for the local homeostatic regulation.
Gastric epithelial cells drive retinoic acid biosynthesis in human gastric dendritic cells (MUC8P.803)
Authors: Bimczok Diane , Beliaeva Olga , Kedishvili Natalia , Smith Phillip , Smythies Lesley
Published: January 2023

Despite the high prevalence of chronic gastritis caused by H. pylori, the gastric mucosa as a unique immune environment has received little investigative attention. Here, we analyzed whether retinoic acid (RA), an important homeostatic factor in small intestine, also contributes to gastric immune regulation. Using HPLC, we detected both RA and the RA precursor molecule retinol in human gastric tissue, indicating that the gastric mucosa is an active site of RA biosynthesis. To determine the source of RA in gastric mucosa, we show that primary human gastric epithelial cells expressed RA biosynthesis genes Rdh10 and Aldh1a1, surprisingly at higher levels than small intestinal epithelial cells. Moreover, primary gastric epithelial cell cultures provided with retinol synthesized RA in vitro and induced the expression of the RA biosynthesis gene Aldh1a2 in co-cultured antigen-presenting cells through an RA-dependent mechanism, evidence that gastric epithelial cells confer the ability to generate RA to gastric DCs. Indeed, DCs purified from gastric mucosa had similar levels of aldehyde dehydrogenase activity and RA biosynthesis gene expression as small intestinal DCs, although gastric DCs lacked CD103. In summary, gastric epithelial cells have access to retinol, convert retinol to RA and drive RA biosynthesis in gastric DCs. Such RA biosynthesis capacity may enable gastric DCs to induce regulatory T cells and drive mucosal T cell homing during H. pylori infection.
Tregitope-mediated antigen-specific tolerance induction in autoimmune and allergic disease in vivo. (THER5P.831)
Authors: Cousens Leslie , Mingozzi Federico , Mazer Bruce , Elyaman Wassim , Khoury Samia , Martin Bill , De Groot Anne
Published: January 2023

Tregitopes are a novel class of therapeutic compounds that harness the activity of regulatory T cells (Tregs) and represent a promising new approach for the treatment of autoimmune and inflammatory diseases. Tregitopes are Treg epitopes found in IgG that provide beneficial immunomodulatory effects, paralleling those attributed to intravenous immunoglobulin (IVIG), in vitro and in vivo. When APCs present Tregitopes to Tregs, APC expression of MHC II, CD80, and CD86 are decreased; expression of the tolerance-associated marker ILT3 is increased. These results are consistent with reported effects of IVIG (Bayry et al. Blood, 2003, 101:758) and the IgG-derived peptide hCDR1 (Sela et al. Immunology, 2009, 128:395). Tregitopes also cause CD4+CD25+FoxP3+ Treg to expand and produce IL-10 in vitro. Evaluation of Tregitope effects in mouse models of MS (EAE), OVA-induced allergic airway disease, and AAV-mediated gene transfer show that effector T cells are modified in the presence of Tregitopes. In OVA-induced allergic airway disease, we observed significant and reproducible expansion of Tregs in conjunction with decreased airway reactivity comparable to, if not greater than, IVIG. We have additional unpublished evidence demonstrating the antigen specificity of tolerance induction using Tregitopes in conjunction with target antigens. Formulated with or without the target antigen, Tregitopes represent a promising new treatment for human immune-mediated disease.
Tussilagone inhibits dendritic cell functions via up-regulating heme oxygenase-1 expression (THER5P.833)
Authors: Park Yunsoo , Ryu Hwa Sun , Kim Ji Sung , Kim Ju Young , Kim Yong Guk , Pyo Min Ji , Park Mi Jeong , Lee Hong Kyung , Hwang Bang Yeon , Kim Youngsoo , Han Sang-Bae
Published: January 2023

Sesquiterpenoid tussilagone (TUS) has a variety of pharmacological activities. In this study, the effects of TUS on dendritic cell (DC) functions and the underlying mechanisms were investigated. TUS inhibited lipopolysaccharide (LPS)-induced activation of DCs, which was proved by the decreases of surface molecule expression, cytokine production, antigen uptake, migration, and allogenic T cell activation. TUS inhibited LPS-induced activation of NF-kB, MAPKs, and IRF-3 signalings. However, TUS did not directly inhibit kinase activities of IRAK4, IRAK1, TAK1 and IKK, suggesting that signaling molecules downstream from toll-like receptor (TLR) 4 might not be the direct targets of TUS. Instead, TUS activated heme oxygenase-1 (HO-1), which was a rate-limiting enzyme involved in heme degradation to immunosuppressive products, such as carbon monoxide and biliverdin. HO-1 antagonist reversed the inhibitory activity of TUS in DCs. Taken together, this study suggests that TUS might be an inducer of HO-1, which indirectly inhibits TLR signalings and functional activation of LPS-treated DCs
Assessment of B cell depletion in combination with IL-2/anti-IL-2 mAb complexes in experimental arthritis (THER5P.824)
Authors: Olalekan Susan , Cao Yanxia , Finnegan Alison
Published: January 2023

Autoimmunity is often associated with a deficiency in functional CD4+CD25+Foxp3+ T regulatory cells (Tregs). In our mouse model of rheumatoid arthritis (RA), proteoglycan induced arthritis (PGIA); we have shown that B cell depletion using a monoclonal antibody against CD20 suppresses disease along with an increase in functional Tregs. In an effort to enhance these Tregs we combined anti-mCD20 with administration of IL-2/anti-IL-2 mAb complexes and investigated the effects of the combination on PGIA. We show in G1 primed mice that IL-2/anti-IL-2 mAb complexes increase the percentage and numbers of Tregs higher than anti-mCD20 or control ab. To determine the effects of the complex on arthritis, we induced PGIA in BALB/c mice. Mice treated with the complex displayed an accelerated severe disease as compared to B cell depleted or control ab treated mice. This was due to an early increase in antigen specific CD4+T cells. Mice that received anti-mCD20 together with IL-2/anti-IL-2 mAb however displayed a less severe disease as compared to the group that only received the complex. Anti-mCD20 treated mice displayed the least arthritic severity. To determine the suppressive capacity of Tregs from the different treatment groups we setup Treg suppression assays. Tregs isolated from B cell depleted mice displayed the highest suppressive capacity. Our findings demonstrate that B cell depletion generates better Tregs and ameliorates PGIA better than IL-2/anti-IL-2 mAb complexes.
Specific HDAC6 inhibition by ACY-738 reduces SLE pathogenesis in NZB/W mice (THER6P.858)
Authors: Regna Nicole , Chafin Cristen , Hammond Sarah , Caudell David , Reilly Christopher
Published: January 2023

Histone deacetylase inhibitors are important modifiers of gene and protein expression. In our studies, we sought to determine if a specific HDAC6 inhibitor (ACY-738) was able to decrease disease in NZB/W lupus-prone mice through regulation of B and T cell differentiation. From 22 - 38 weeks-of-age, NZB/W mice were injected intraperitoneally with 20 mg/kg of ACY-738, 5 mg/kg of ACY-738, or DMSO (vehicle control). Body weight and proteinuria were measured every 2 weeks, while sera anti-dsDNA, Ig isotypes, and cytokine levels were measured every 4 weeks. Kidney disease was determined using sera and urinary markers of SLE, immune complex deposition, and renal pathology. Flow cytometric analysis was used to assess thymic and splenic T cell profiles as well as bone marrow, splenic, and peripheral B cell differentiation patterns. Our results showed that HDAC6 inhibition was able to decrease many hallmarks of SLE disease including splenomegaly, immune complex-mediated glomerulonephritis, sera anti-dsDNA levels, and inflammatory cytokine production. ACY-738 treatment decreased the number of double-negative thymic T cells and increased the percentage of splenic Treg cells. Inhibition of HDAC6 altered BM B cell differentiation by increasing the percentage of cells in the early-stage developmental fractions of both pro-and pre-B cells. These results suggest that specific HDAC6 inhibition may be able to decrease SLE disease by altering aberrant T and B cell differentiation.
A novel concept for tolerization of diabetogenic CD4+ T cells by intranasal treatment with CTA1R7K-peptide-DD (THER6P.859)
Authors: Cardell Susanna , Langenes Veronica , Fahlén-Yrlid Linda , Löfbom Linda , Lycke Nils
Published: January 2023

Type 1 diabetes results from CD4+ T cell dependent immune destruction of insulin producing β-cells in the pancreas islets. Tolerization of diabetogenic CD4+ T cells would therefore be an important advancement in the development T1D treatments. We previously described that a mutated (R7K), enzyme killed, cholera toxin A1 subunit based adjuvant CTA1R7K-DD induced specific tolerance rather than enhancement of immunity, and moreover, ameliorated murine collagen induced arthritis. Here, we investigate tolerization of diabetogenic T cells of the non-obese diabetic NOD mouse, exploring diabetogenic TCR transgenic BDC2.5 CD4+ T cells. Treatment of BDC2.5 NOD mice intranasally with CTA1R7K-PS3-DD fusion protein (PS3 = BDC2.5 TCR mimotope peptide) reduced proliferation and IFN-gamma production to PS3 peptide restimulation. Transferred CD4+ BDC2.5 T cells induced diabetes in 80-100% of recipient NOD.scid mice. In contrast, if either donor BDC2.5 NOD mice, or recipient NOD.scid mice, had been treated with CTA1R7K-PS3-DD the majority of mice remained healthy. The i n treatment resulted in systemic increase in FoxP3+ CD4+ BDC2.5 T cells, most enhanced in draining mediastinal lymph nodes. However, transfer of FoxP3 negative CD4+ BDC2.5 T cells from CTA1R7K-PS3-DD treated mice still demonstrated reduced diabetes induction compared to cells from control treated mice, suggesting that tolerance in the recipient mice is not depending on the action of FoxP3 Treg cells.
Targeting the JAK/STAT pathway in the treatment of Parkinson’s disease (THER6P.847)
Authors: Qin Hongwei , Buckley Jessica , Liu Yudong , Holdbrooks Andrew , Benveniste Etty (Tika)
Published: January 2023

Parkinson’s Disease (PD) is an age-related, chronic neurodegenerative disorder. At present, there are no therapies to prevent PD progression. Activated microglia and subsequent neuroinflammation associate with the pathogenesis and progression of PD. Accumulation of α-synuclein (α-syn) in the brain is a core feature of PD and leads to microglial activation, inflammatory cytokines/chemokines production and neurodegeneration. Given the importance of the JAK/STAT pathway in activating microglia and inducing cytokine/chemokine expression, we investigated the therapeutic potential of inhibiting the JAK/STAT pathway using the JAK1/2 inhibitor, AZD1480. We utilized an in vivo rat model of PD induced by viral overexpression of α-syn. We find that AZD1480 treatment inhibits α-syn-induced neuroinflammation by inhibiting microglia activation and CD3+ T-cell infiltration, and neurodegeneration by preventing the degeneration of dopaminergic neurons in vivo. In vitro, AZD1480 inhibits α-syn- and IFN-γ-induced MHC Class II, CD40, iNOS, TNF-α and IL-6 expression in microglia and macrophages by reducing STAT1 and STAT3 activation. These results indicate that inhibiting the JAK/STAT pathway can prevent neuroinflammation and neurodegeneration by suppressing activation of innate and adaptive immune responses to α-syn and inflammatory cytokines in this PD model. Furthermore, this suggests the feasibility of targeting the JAK/STAT pathway as neuroprotective therapy for neurodegenerative diseases.
Dysregulated cytokine production by leukocytes, mediated by SOCS1 deficiency, is correlated to skin pathology: implications for therapeutic targeting. (THER6P.851)
Authors: Lam Brandon , Wilson Tenisha , Bedoya Simone , Armbruster Cristina , Johnson Howard , Larkin Joseph
Published: January 2023

Dysregulated leukocyte activation and cytokine signaling are known to contribute to autoimmunity. Notably, although Suppressor of Cytokine Signaling-1(SOCS1) is important in the regulation of both leukocyte activation and cytokine signaling, how deficiencies in SOCS1 relate to autoimmunity is not well understood. Therefore we analyzed leukocytes from mice bearing heterozygous expression of SOCS1 (SOCS1+/-) for dysregulated immune function. LPS stimulated SOCS1+/- leukocytes exhibited enhanced production of IL6, and IL12. Notably we observed that SOCS1+/-IFNγ-/- mice, but not IFNγ-/-, SOCS1+/-, or wild-type mice exhibited significant skin pathology on the back and ears. The observed skin pathology in SOCS1-/-IFNγ-/- mice was correlated to leukocyte accumulation within draining lymph nodes and spleen. In vitro, SOCS1+/-IFNγ-/- leukocytes activated with αCD3/αCD28 were hyper-proliferative and produced higher levels of IL17 when compared SOCS1+/+ littermates. Finally, a peptide capable of partially mimicking SOCS1 (SOCS1-KIR) was sufficient to reduce excessive proliferation, and aberrant IL12 and IL6 production of SOCS1+/- leukocytes in vitro. Together these results implicate an important role of SOCS1 in the regulation of immune responses. Moreover these results suggest that targeting SOCS1 deficiency, by a mimetic of SOCS1, may have implications in the regulation of skin pathologies mediated by immune dysregulation.
Immunomodulatory effect of pentoxifylline and stored blood on neutrophil functions (TRAN3P.862)
Authors: Abdelrahman Zakia , Abou-Shamaa Lobna , Sadek Nadia , El hadidy Abeer , Kabary Bassam
Published: January 2023

Background: Allogeneic blood transfusions expose a patient to many soluble and cell-bound antigens, expressed on viable and decaying cells. Blood transfusion is a risk factor for many inflammatory processes. Its supernatant fraction has been proven to activate neutrophils Objective: To evaluate the immunomodulatory effects of stored blood supernatant and pentoxifylline on neutrophil functions Methods: Neutrophils(N) of recipient blood samples were incubated with supernatant stored blood (35 days blood bags) (N+S)or fMLP(N+F) or(N+S+F).Neutrophils incubated with PTX (N+P),(N+S+P), (N+F+P) and (N+S+F+P) .Also neutrophil functions studied by CD11b expression, phagocytosis, respiratory burst and apoptosis Results: Neutrophil expression of CD11b ,phagocytic activity, neutrophil intracellular superoxide anion production and neutrophil apoptosis in presence of (N+F), (N+S) and (N+F+S) were significantly increased in comparing to baseline. Combined incubation with (N+ F + S) induced significant increase in neutrophil functions than (N+F) or (N+S). PTX significantly attenuated neutrophil functions induced by (N+F), (N+S), ( N+S+F) Conclusion: Stored blood supernatant has an immunomodulatory effects on neutrophil function(priming them) through CD11b expression, phagocytosis, respiratory burst and apoptosis. PTX downregulates these neutrophil functions. PTX may have therapeutic potential in inflammatory injury due to blood transfusion.
Analysis of islet transplant infiltrate T cells informs design of therapeutic strategies which promote long-term euglycemia (TRAN3P.871)
Authors: Burrack Adam , Nelsen Michelle , Coulombe Marilyne , Kupfer Tinalyn , Gill Ronald
Published: January 2023

NOD mice, a well-characterized model of type 1 diabetes, provide a stringent model in which to test transplant tolerance-promoting therapies. We studied the failure of T cell-directed therapies in the autoimmune recipient. When we blocked T cell co-stimulation with a CD154 antibody in NOD isograft recipients transplant survival time doubled (MST 22 days), and we observed a profound decrease in ex vivo cytokine production of graft infiltrating CD4+ T cells. These results suggest that autoimmune CD8+ T cells can mediate disease recurrence with minimal help from CD4+ T cells and that CD154 antibody does not impede the reactivation of primed autoreactive CD8+ T cells. Graft survival was significantly prolonged in NOD mice grafted with syngeneic islets and the combination treatment of CD154 antibody and transient CD8+ T cell depletion (MST 58 days). In the clinically relevant model of islet allografts in NOD mice, anti-CD154 antibody treatment had less effect on graft rejection (MST 10 days), and minimal effect on ex vivo CD4+ T cell cytokine production. Unfortunately, our combination therapy demonstrated minimal allograft survival benefits (MST 12 days). Interestingly, CD4+ T cell depletion therapy yielded superior allograft survival results (MST 24 days). These results highlight clinically relevant differences in the mechanisms of autoimmune disease recurrence and islet allograft rejection by the autoimmune recipient.
Effect of systemic anti-154 (CD40L) antibody on clinical outcomes of full-thickness porcine corneal graft in pig-to-rhesus corneal transplantation model (TRAN3P.872)
Authors: Kim Dong Hyun , Choi Hyuk Jin , Kim Mee Kum , Lee Hyun Ju , Jeong Hyun Jeong , Ryu Jin Suk , Kang Hee Jung , Park Chung-Gyu , Wee Won Ryang
Published: January 2023

Purpose: To investigate the effect of systemically administered anti-CD154 antibody(Ab) on the survival of full-thickness porcine corneal grafts. Methods: Seven rhesus macaques were assigned to control (n=3) or immunosuppression (n=4) group and underwent full-thickness corneal transplantation. Systemic steroid was applied to control group while anti-CD154 Ab were administered to immunosuppression group. Rejection sign, central corneal thickness (CCT), and recipients’ immunologic profile including memory T cell, anti-α-Gal and donor-specific Abs, mixed lymphocyte-like reaction and aqueous complement concentration were evaluated. Cellular infiltration into the xenografts was evaluated by immunohistochemical staining. Results: Anti-CD154 Ab-based immunosuppression significantly prolonged the survival of full-thickness xenocorneal grafts. In control group, CCTs, memory T cells, anti-α-Gal IgG and donor pig-specific IgG Abs continuously increased in correspond with the progression of graft rejection in contrast to immunosuppression group. Reactivities to donor-specific stimulators and concentrations of the aqueous complement were also significantly higher in control group than in immunosuppression group. Rejected grafts showed extensive cellular infiltration. Conclusions: Anti-CD154 antibody-based immunosuppression is effective for prolonging the survival of full-thickness xenocorneal grafts and controlling immunologic responses in pig-to-rhesus corneal transplantation model.
Pregnancy primes anti-fetal T cell memory capable of allograft destruction (TRAN3P.874)
Authors: Porrett Paige , Barton Brendan , Klahr Rebecca , Wherry John
Published: January 2023

Pregnancy-induced anti-fetal antibody can destroy allografts and excludes many women from transplantation, but anti-fetal T cell responses are poorly understood and are not currently evaluated in potential organ recipients. To test whether pregnancy (PG) primes graft-reactive T cell memory, we compared Ova skin graft survival (GS) between naive B6 mice (N), mice mated to Ova transgenic (tg) males (Ova PG-sensitized; OP), or mice sensitized by a previous Ova skin graft (OS). Skin grafts were placed 30 days after either Ova pup delivery (OP group) or after rejection of an Ova skin graft (OS group). OP females rapidly rejected Ova skin grafts with second set kinetics identical to OS females [GS (days): 11.3±1.5(OP) vs. 10.3±0.6(OS) vs. 15±0.7(N); p<.01]. To test the effects on antigen-specific T cells, mice were injected with Ova-specific TCR tg T cells. Flow cytometric analysis 30 days after pup delivery revealed memory T cell differentiation in response to fetal antigen [%anti-Ova CD8+CD44+CD62L-: 85%(Ova-mated) vs. 38%(Balb/c-mated)]. OP mice also produced enhanced effector T cells upon secondary challenge with an Ova skin graft [%anti-Ova CD8+IFNγ+: 60%(OP) vs. 43%(Balb-mated) vs. 35%(N)]. These results indicate that PG can prime T cell memory that gives rise to secondary effector T cells that mediate rapid graft destruction. Stimulation of PG-induced anti-fetal T cell memory may contribute to the inferior GS observed in women who receive a transplant from a child or spouse.
Sera miR-338-5p differentially expressed in renal transplant recipients and negatively related with soluble BAFF (TRAN3P.877)
Authors: Xu Haiyan , He Xiaozhou , Ma Xuyi , Xue Dong , Dong Panpan , Zhang Yanyun , Zhang Xueguang
Published: January 2023

To investigate the expression characteristic of sera miR-338-5p in renal transplant (RTx) recipients, and further analyse its potential significance. Serum of RTx recipients (49 cases) were collected. miR-338-5p were extracted and detected by qPCR, soluble B cell activating factor (sBAFF) was determined by ELISA (R&D), and the presence of circulating anti-HLA&MICA was determined by single-antigen flow bead assays (One Lambda) on a Luminex platform. Healthy volunteers (20 cases) were controls. SPSS17.0 software was applied, two-side t-test was to analyse the means of two-independent samples, Spearman method was to analyse the correlation of miR-338-5p and sBAFF, anti-HLA&MICA. P<0.05 was considered to be significant. Compared with healthy controls, miR-338-5p in RTx recipients significantly decreased and sBAFF significantly increased (P<0.05). miR-338-5p significantly down-regulated in RTx recipients within 1-year post-operation, compared with those over 1-year post-operation (P<0.05); miR-338-5p also signficantly down-regulated in recipients within 3-year post-operation, comapred with those over 3-year post-operation (P<0.05). sBAFF level in different period after operation was not significant. For all subjects, miR-338-5p was negatively correlated with sBAFF (r=-0.51, P<0.001), and sBAFF was positively correlated with anti-MICA antibody (r=0.86, P<0.05). miR-338-5p may involve in the procedure of immune response after RTx by indirectly or directly targeting BAFF signal.
miRNA-181a is critical for the tolerogenic properties of plasmacytoid dendritic cells (TRAN3P.886)
Authors: Lau Audrey , Schaffert Steven , Qu Xiumei , Krams Sheri
Published: January 2023

Liver allografts are well tolerated and other solid organ allografts, when transplanted concurrently with livers, show improved graft outcomes. However, the mechanisms underlying “hepatic tolerance” have yet to be elucidated. Previous data show that hepatic dendritic cells (HDC) have diminished antigen presenting and immune stimulatory function compared with DC in lymphoid tissue. It has been hypothesized that immature plasmacytoid (p)DC are inherently tolerogenic with studies supporting this hypothesis. Our work confirms that HpDC prolong cardiac allograft survival (p<0.01) in a murine model compared to bulk and myeloid (m)DC. Micro-ribonucleic acid (miR) are short (15-22 nucleotides) RNA molecules which can post-transcriptionally regulate messenger RNA transcripts, resulting in translational repression or upregulation. We hypothesize that miR-181a, which we found is highly expressed in HpDC, plays a critical role in the tolerogenic properties of pDC. TLR 9 stimulation of pDC from miR-181a-/- mice exhibited higher levels of maturation markers and differential cytokine production as compared to wild-type C57BL/6 mice. More, there is altered phosphorylation of key members of signal transduction pathways, such as ERK1/2. Finally, in the same murine model of cardiac allograft transplantation, use of pDC from miR-181a-/- mice abrograted the tolerogenic properties seen with HpDC. Together, our data shows a critical role for miR-181a in the tolerance-inducing properties of pDC.
Recipient’s monocyte-derived dendritic cells and macrophages play key roles in acute cardiac allograft rejection (TRAN3P.864)
Authors: Divito Sherrie , Liu Qi , Shufesky William , Nakao Atsunori , Rojas-Canales Darling , Wang Zhiliang , Ochoa Juan , Larregina Adriana , Morelli Adrian
Published: January 2023

Donor-derived dendritic cells (DCs) migrating out of allografts into recipient lymphoid organs, and recipient lymphoid-resident DCs within draining lymphoid organs are classically considered the instigators of acute rejection in heart transplants. However, the role of the more recently described CD11cint CD11b+ Ly6C+ monocyte-derived (mo) DCs in acute allograft rejection is unknown. We demonstrate in a mouse model that cardiac allografts are infiltrated by recipient mo-DCs and mo-macrophages, each originating from different subsets of blood monocytes, within one week of transplantation. Graft-infiltrating mo-DCs are the main producers of IL-12p70 and exhibit potent T cell allo-stimulation and Th1 bias. Ablation of DCs after allo-sensitization correlates with reduced T cell proliferation and increased T cell death inside the graft, and prolonged transplant survival. Comparatively, graft-infiltrating mo-macrophages are far less efficient Ag-presenting cells, but release TNF-α and nitric oxide, the latter of which impairs T cell survival and promotes allograft survival. Our results indicate that both recipient mo-DCs and mo-macrophages play key roles during acute rejection within heart allografts, and that these cell subtypes, or potentially their blood monocyte precursors, could serve as therapeutic targets to prevent or treat acute rejection.
Effector T cells adopt fatty acid metabolism in response to environmental factors present at the time of activation (TRAN3P.898)
Authors: Byersdorfer Craig , Tkachev Victor , Goodell Stefanie , Swanson Jacob , Ferrara James
Published: January 2023

Lymphocyte metabolism following in vivo activation remains poorly understood. Compared to naïve cells, allogeneic T cells activated during graft-versus-host disease (GVHD) increased fatty acid (FA) transport (44.3 ±6.9% vs. 0.7 ±0.1%, p=0.003), up-regulated FA oxidation enzymes (e.g. carnitine palmitoyltransferase 1a (CPT1a)), oxidized 3-fold more fat ex vivo (p=0.0001), and increased levels of PGC-1α, an oxidation co-activator (0.9 ±0.2 vs. 0.03 ±0.01, p=0.005). We next examined whether environmental factors influenced a T cell’s metabolic adaptation. OT-I T cells were transferred into irradiated CAG.OVA mice, to recapitulate GVHD, or into naïve C57Bl/6 mice challenged one day later with CAG.OVA dendritic cells (i.e. cellular immunization). Cells proliferated robustly in both models (>10 times by day 7), but only during GVHD did OT-I cells escalate FA transport (40.0 ±6.5% vs. 9.0 ±3.0%, p=0.0002) and up-regulate levels of PGC-1α (1.0 vs 0.09 (p = 0.0004)). Furthermore, inhibiting FA oxidation during GVHD decreased allogeneic T cells by 35% (5.3 ±1.5x10^6 vs.3.4 ±1.2 x10^6, (p=0.01)) and improved GVHD clinical scores, without affecting syngeneic reconstitution. In total, these data demonstrate that effector T cells adopt FA metabolism in response to environmental cues present at the time of activation (e.g. ubiquity of antigen) and we conclude that inhibiting FA oxidation may be a selective means to eliminate pathogenic T cells while preserving homeostatic immune responses.
Targeting sirtuin-1 improves murine renal allograft survival and function (TRAN3P.901)
Authors: Beier Ulf , Wang Zhonglin , Xiao Haiyan , Bhatti Tricia , Hancock Wayne , Levine Matthew
Published: January 2023

Pharmacologic inhibition of sirtuin-1 (Sirt1) augments suppressive functions of Foxp3+ regulatory T cells (Treg) and prolongs murine cardiac allograft survival. We investigated if Sirt1 targeting produced longer allograft function and allograft-dependent survival in a MHC-mismatched renal transplant model. After transplantation of BALB/c kindey allografts into C57Bl/6 wild type or fl-Sirt1/CD4cre recipients, native kidneys were removed three days post-operatively. We followed the mice for survival, and allograft function by weekly serum electrolyte and renal function (BUN, creatinine), as well as hematocrit. We observed, that loss of Sirt1 in T cells led to prolonged median renal allograft-dependent survival from 14 days (IQR: 11-91.25) to 143 days (IQR: 27-147) in wild type controls vs. fl-Sirt1/CD4cre recipients (Mantel-Cox, p<0.0004). In addition, by comparing mice that survived until 13 weeks, loss of Sirt1 in the recipient’s T cells led to improved renal allograft function, with 34.2 ±20 vs. 90.8 ±37.6 mg/dL BUN (p<0.001), and lower creatinine 0.33 ±0.14 vs. 0.61 ±0.3 mg/dL (p<0.001) in fl-Sirt1/CD4cre vs. wild type control mice, respectively. Long-term surviving fl-Sirt1/CD4cre renal transplant recipients (>100 days) became tolerant to antigen re-challenge with a BALB/c, but rejected third-party C3H cardiac allografts. In conclusion, deletion of Sirt1 in T cells is effective at prolonging allograft function and allograft-dependent survival.
Intrathymic innate lymphoid cells: long-lived mediators of immune regeneration (TRAN3P.905)
Authors: Dudakov Jarrod , Yim Nury , Cole Denzel , Mertelsmann Anna , O'Connor Margaret , Jenq Robert , Velardi Enrico , Young Lauren , Hanash Alan , van den Brink Marcel
Published: January 2023

Innate lymphoid cells (ILC) are a newly described population of immune cells implicated in the maintenance and function of tissues as diverse as liver, gut, lung and lymph nodes. We have recently described a key role for ILC, and their production of IL-22, in endogenous thymic regeneration: a crucial process that allows for renewal of immune competence following immune depletion. Unlike other lymphoid cells, ILC were extremely radio-resistant with little depletion of cells after even lethal doses of total body irradiation. Consistent with these findings, a considerable proportion of ILC were non-cycling in steady-state conditions and expressed high levels of the anti-apoptotic protein Bcl-2. Perhaps unsurprising given their resistance to proliferation-targeted damage, a residual population of host ILC could be identified for up to 12 months after hematopoietic stem cell transplantation and these host ILC were almost exclusively non-proliferating. Intriguingly, although IL-22 appears to play a considerable role in their regenerative capacity, depletion of ILC from IL-22 deficient mice led to significantly worse recovery compared to Il22-/- mice replete with ILC. These pre-clinical studies not only help to identify the mechanisms that allow this nascent cell population to mediate its regenerative effects, but also offer a novel clinical approach to enhance T cell immunity in individuals with deficiencies due to aging, infectious disease, chemotherapy or radiation injury
Human IL-10 exacerbates graft versus host disease in NOD-scid IL2rγcnull mice transplanted with human PBMCs (TRAN3P.910)
Authors: Abraham Sojan , Choi Jang Gi , Manjunath N. , Shankar Premlata
Published: January 2023

Dysregulated cytokine production plays an important role in the clinical manifestation of acute graft-versus-host disease (aGVHD) following allogeneic bone marrow transplantation (BMT). The role of IL-10, anti-inflammatory cytokine in GVHD pathogenesis remains ambiguous. There are reports showing both beneficial and adverse effects of this cytokine in GVHD progression. Hence to determine the role of IL-10 in GVHD progression, we expressed hIL-10 in NOD-scid IL2rγcnull mice (NSG) by hydrodynamic gene delivery and evaluated its effect on xeogenic GVHD manifestations. We demonstrate that hIL-10 is capable of accelerating the hPBMC induced xeno-GVHD in NSG mice. The marked human T cell infiltration and tissue destruction observed in presence of IL-10 suggest that human IL-10 potently activates and expands pathogenic human T cells with the capacity to induce xeno GVHD. The significant reduction in the number of human cells at the initial phase and robust expansion at later stage suggests that those escapes IL-10 mediated suppression is responsible for the development of xeno-GVHD. Taken together, our results demonstrate for the first time that xenografted human T cells expand in response to IL-10 and mediate tissue damage in xeno GVHD mouse model. These finding provide caution against using rhIL-10 as an immunosuppressive cytokine after allogenic BMT.
Asymptomatic ocular herpes infections precipitate rapid rejection of corneal allografts (TRAN3P.917)
Authors: Rowe Alexander , Yun Hongmin , Hendricks Robert
Published: January 2023

Most herpes simplex virus type 1 (HSV-1) infected individuals are asymptomatic despite harboring latent virus in their trigeminal ganglia (TG). To determine if these ‘quiet’ infections influence corneal transplant survival we infected the corneas of C57Bl/6 mice with an HSV-1 strain that causes a latent TG infection but no overt corneal pathology. Infected mice received a corneal transplant from syngeneic mice that express ovalbumin (OVA) as a surrogate alloantigen. Graft survival was 9% in infected mice as compared to 58% in non-infected mice. Transcripts for chemokines including CXCL10, CXCL9, CCL5, and CCL2 were significantly elevated, and were significantly reduced by CD4 T cell depletion prior to infection. To assess CD4 T cell antigen specificity irradiated wild type C57BL/6 mice were reconstituted with mixed BM from CD4 KO mice and RAG-/- OT-II transgenic mice so all CD4 T cells were OVA-specific. The chimeras were not infected or received a quiet HSV-1 infection with or without transfer of polyclonal CD4 T cells prior to transplantation of OVA expressing grafts. Graft survival in chimeras was: 100% (non-infected), 60% (quiet infection, no T cell transfer), and 20% (quiet infection with CD4 T cell transfer). We conclude that quiet HSV-1 corneal infections can augment CD4 T cell mediated corneal allograft rejection by increasing chemokine expression in the graft bed and possibly recruiting HSV-specific CD4 T cells into the rejection response.
Autoimmunity as an endogenous source of heterologous alloimmunity (TRAN3P.868)
Authors: Burrack Adam , Nakayama Maki , Landry Laurie , Coulombe Marilyne , Gill Ronald
Published: January 2023

Curiously, although autoimmunity is restricted to self MHC molecules, spontaneously diabetic NOD mice reject MHC-unrelated islet allografts more rapidly than syngeneic (NOD) grafts. We hypothesized that the autoimmune (islet-reactive) T cell repertoire harbors a subset of TCRs that also cross-react to allogeneic MHC molecules. To test this concept, we performed high throughput TCR sequencing of T cells infiltrating MHC-mismatched (C3H, H-2k) islet allografts on the day of rejection in diabetic female NOD mice. TCRs expressed in high frequency in the allograft (>1%) were cloned and tested for islet autoreactivity and/or alloreactivity following TCR cDNA transfection into IL-2 producing hybridoma cells. Of note, three TCRs studied that were the most frequent in the graft were also detected in the endogenous pancreas. All three transfectants expressing these TCRs were islet reactive. Importantly, all three of these TCR also were directly alloreactive to C3H spleen cells. Two of the three TCRs were MHC class I reactive (anti-H-2Dk), and the other was MHC class II-reactive (anti-I-Ak). These results suggest that dual auto/allo-reactive T cells are selectively expanded in islet allografts. As such, we propose that autoimmunity is an unusual source of endogenous heterologous alloreactivity that may accelerate allograft rejection by simultaneous auto- and allo-reactivity.
In-vivo testing of PSMA-targeted T-cell immunotherapy for prostate cancer (TUM7P.920)
Authors: Liu Zhe , Papa Sophie , Morris Jonathan , Maher John
Published: January 2023

Introduction: Skeletal metastases are a significant cause of morbidity and mortality and overall greatly affect the quality of life of prostate cancer patients. Examining impact of bone metastasis in vivo remains a significant challenge, as animal models that closely mimic the natural history and malignant progression of clinical prostate cancer are not available. Objectives: To develop an animal model of human metastatic prostate cancer. Once a model has been developed and optimized, it was this to test efficacy of immunotherapy using T-cells that have been genetically targeted against prostate-specific membrane antigen (PSMA). Material and methods: PCR, western blot, flow cytometry and ELISA, we performed functional anaylsis of fucosyltransferase 3(FT3) in PC3LN3(PL)and PC3LN3-PSMA (PLP) tumour cell lines. In vivo bioluminescent imaging (BLI) was used to detect metastases. Results: In preliminary studies, we have observed that delivery of a FT3-encoding retroviral vector to PL and PLP enables them to express sialyl Lewis X and to acquire E-selectin binding activity. We also showed that FT3 promotes increased PLP motility and invasiveness in vitro. Bioluminescent animal model of metastasised prostate cancer is established to determine the effect of this upon their pattern of metastatic spread in SCID Beige mice. Conclusion: We have established an in-vivo model of PSMA-expressing prostate cancer. This will serve as a platform to test immunotherapy using P28z+ T-cells.
VCAM-1 enhances immune evasion in a murine model of metastatic cervical cancer (TUM7P.938)
Authors: Huang Alex , Scrimieri Francesca , Tong Alexander , Askew David , Eid Saada , Bobanga Iuliana , Corn David
Published: January 2023

Surface over-expression of Vascular Cell Adhesion Molecule-1 (VCAM-1) was shown to provide a pro-survival advantage in metastatic breast cancer. Similarly, the immune-resistant murine cervical cancer A17 expresses an increased surface VCAM-1 expression compared with the non-metastatic parental tumor, TC1-P0. To test whether VCAM-1 overexpression was responsible for the immune evasive phenotype in A17, we modulated surface VCAM-1 levels on A17 using an shRNA knockdown approach. We inoculated these cells intravenously and subcutaneously into both C57BL/6 and athymic nude mice to interrogate how VCAM-1 affects surrounding immune cell interactions with the tumor, including the use of intravital 2-photon microscopy. VCAM-1lo A17 exhibited reduced growth in C57BL/6 mice but had similar growth kinetics as wild type A17 in nude mice, suggesting an extrinsic T-cell dependent role for tumor-associated VCAM-1 that was responsible for the differences in vivo. Moreover, VCAM-1lo cell lines showed decreased lung metastasis in both immune-competent mice and athymic nude mice, coinciding with changes in M1/M2 polarized macrophages and altered cytokine productions. Together, our results define a functional role for tumor-associated VCAM-1 in helping aggressive tumors circumvent antitumor immune surveillance, and support VCAM-1 as a target for novel immunotherapy against metastatic cervical cancer.
Sensitization of BRAFi- and immune- dual resistant melanomas to immune effector cells through HDACi-mediated regulation of apoptotic machinery (TUM7P.940)
Authors: Jazirehi Ali , Nazarian Ramin , Torres-Collado Antonio , Economou James
Published: January 2023

BRAFV600E-inhibitors (BRAFi; e.g., vemurafenib) and modern immune-based therapies such as PD-1/ PD-L1 and CTLA-4 checkpoints blockade and adoptive cell transfer (ACT) have significantly improved the care of melanoma patients. Having these two effective therapies raises the question whether there is a rational biological basis for using them in combination. We developed an in vitro model to determine whether tumor resistance mechanisms to a small molecule inhibitor of a driver oncogene, and to cytotoxic T lymphocyte (CTL)- and natural killer (NK) cell-delivered death signals were exclusive or intersecting. We generated melanoma sublines resistant to BRAFi vemurafenib and to CTL recognizing MART-1 melanoma antigen. Vemurafenib-resistant (VemR) sublines were cross-resistant to MART CTL and NK cells indicating a common apoptotic pathway governing tumor response to both modalities was disrupted. Pretreatment of VemR melanomas with a histone deacetylase inhibitor restored sensitivity to MART CTL and NK apoptosis by skewing the apoptotic gene programs towards a proapoptotic phenotype. Our findings suggest that during the course of acquisition of BRAFi resistance, melanomas develop cross-resistance to CTL- and NK-killing. Further, aberrant apoptotic pathways, amenable by an FDA-approved chromatin modifier, regulate tumor resistance mechanisms to immune effector cells. These results provide rational molecular basis for further investigations to combine these therapies clinically.
Costimulatory effects of TLR1-TLR2 stimulation on CD8+ T cells is dependent on 4-1BB signaling (TUM7P.941)
Authors: Joseph Ann , Srivastava Ratika , Zabaleta Jovanny , Kaczanowska Sabina , Davila Eduardo
Published: January 2023

Toll-like receptors (TLRs) play a significant role in amplifying immune responses against tumors. Our studies demonstrate that activated CD8+ T cells express TLRs and upon stimulation with TLR1-TLR2 agonists, exhibit enhanced T cell survival and proliferation. To define how TLR signaling promotes these processes, we compared gene expression analysis between TLR1-TLR2-stimulated and unstimulated CD8+ and MyD88-/- CD8+ T cells and identified significant increases in the expression levels of five TNF family members. We sought to test the hypothesis that the costimulatory effects of TLR1-TLR2 stimulation on CD8+ T cells were dependent on the function of one of these costimulatory molecules. Blocking CD137 (4-1BB) using blocking antibodies, but not the other TNF family members, dramatically reduced the costimulatory effects of TLR-mediated T cell proliferation. Likewise, 4-1BB -deficient CD8+ T cells did not respond to TLR stimulation. While TLR signals are capable of upregulating antiapoptotic proteins such as BCL2 and BCL-xL, mechanistically, we postulate that 4-1BB activation stabilizes these proteins, hence promoting survival. Adoptive transfer of pmel T cells along with the 4-1BB agonistic antibody, 3H3 and the TLR ligand, Pam3CSK4 was able to reduce tumor growth in a B16 melanoma model. These studies indicate that the costimulatory effects of TLR1-TLR2 signaling in CD8+ T cells mediated by 4-1BB signals are vital for mounting an effective antitumor response.
Enhancing the antitumor effects of adoptive cell transfer immunotherapy using preconditioning thermal ablation (TUM7P.956)
Authors: Bucsek Mark , Ito Fumito , Muhitch Jason , Ajith Ashwin , Evans Sharon
Published: January 2023

A major challenge facing T cell based immunotherapeutic strategies is the limited trafficking of cytotoxic T cells at vascular checkpoints within the tumor microenvironment. Here, we report the use of radiofrequency ablation (RFA; 90°C, 1min), a cytoreductive strategy used clinically to treat unresectable tumors, as a pre-conditioning regimen to overcome deficient CD8 T trafficking across tumor vasculature. RFA pre-treatment caused a concomitant decrease in the number of Foxp3+CD4+CD25+ regulatory T cells while bolstering CD8 T cells in tumor draining lymph nodes and the non-ablated region within the tumor microenvironment. Taking advantage of the immunostimulatory activities of RFA, adoptive transfer of antigen-specific T cells was performed 6 hours post-RFA in implantable murine tumors (B16.F10 melanoma or CT26 colorectal tumors). Increased trafficking of adoptively transferred T cells was observed in both the tumor draining lymph node and tumor microenvironment. Improved trafficking was associated with a robust increase in antigen-restricted apoptosis of tumor cell targets and significantly enhanced overall survival without recurrence. Further investigation revealed that adoptive T cell transfer following RFA elicited a durable tumor-specific memory response. Taken together, these findings suggest that thermal ablative regimens may have clinical potential as an adjuvant to augment the efficacy of T cell-based cancer immunotherapies.
HBP1, a novel protein from lamprey can repress proliferation of cancer cells (TUM7P.926)
Authors: Li Qingwei , Pang Yue , Huang Jinsha , Liu Shuang
Published: January 2023

HBP1 ( HMG-box containing protein 1) is homologous to the sequence- specific high mobility group ( HMG) family of transcriptional factor. Previous work had shown that HBP1 inhibits G1 progression in cells. In this paper, we identified a homolog of HBP1 in the Japanese lamprey (Lampetra japonica). The bioinformatics analyses showed that the Lj-hbp1 gene has over 60% sequence identity with its homologs in jawed vertebrates. The open reading frame (ORF) of Lj-HBP1 is 1584 bp long and encoded 527 amino acids. The calculated molecular masses of Lj-HBP1 was 58 kD. Real-time quantitative PCR indicates that the HBP1 appears universally in the lampreys tissues. An Adeno-X-LjHBP1 vector were constructed, HBP1 transcription factor were expressed, and its infection efficiency of the packaged lentivirus were evaluated by PCR method. We found that both human tumor cells and normal cells were infected efficiently as determined by the expression of HBP1. In addition, the proliferation rate and ability of growth in soft agar of the HBP1-transfected Raji cells were suppressed through growth curve test ( population doubling as index) and soft agar assay. The results indicated that modest overexpression of HBP1 inhibited the growth of tumor cells.
Folate-modified chitosan nanoparticles coating IP-10 promote pMAGE-A1278-286 specific CTL responses to liver cancer (VAC11P.1011)
Authors: Lu Xiaoling , Zhao Yongxiang , Su Jing , Zhou Sufang , He Jian , Lai Chunhui , Li Xi , Duan Siliang
Published: January 2023

Adoptive therapy using tumor antigen-specific cytotoxic T lymphocytes (CTLs) is a promising approach for the treatment of human cancers. However, tumor antigen-specific CTLs are difficult to reach tumor tissues because of immune suppressions in cancer patients. Interferon-inducible protein-10 (IP-10), a powerful chemokines, effectively attracts CTLs to tumor tissues and improves the anti-tumor activity of CTLs. Increasing the expression of IP-10 in tumor tissues can efficiently promote the efficacy of adoptive therapy. In this study, we developed folate-modified chitosan nanoparticles coating the human IP-10 gene (FA-CS-hIP-10), that specifically bound to the folate receptors on hepatoma cells and promote the expression of IP-10 to improve the activity of pMAGE-A1278-286 specific CTLs. Combination of FA-CS-hIP-10 and pMAGE-A1278-286 specific CD8+ CTLs efficiently increased the secretion of IFN-γ, inhibited the growth of tumor and extend the survival of nude mice subcutaneously transplanted human liver cancer. Our results demonstrated that the mechanism of this novel adoptive therapy involved in the inhibition of angiogenesis, inhibition of proliferation, and promotion of apoptosis of tumor cells. Our study provided a potentially novel approach for the treatment of human liver cancers by improving the activity of tumor antigen-specific CTLs.