bluuebunny/crossref_metadata_2025 huggingface dataset:
Tregitope applications to tolerance induction in autoimmune diseases. (116.10)
Authors: Cousens Leslie , De Groot Anne , Najafian Nader , Mingozzi Federico , Elyaman Wassim , McClaine Elizabeth , Mazer Bruce , Hui Daniel , Khoury Samia , Su Yan , Scott David , Martin William
Published: January 2023

Modulation of T cell responses may contribute to the design of new approaches for the treatment of autoimmune and inflammatory diseases. IVIG is one example of a therapy with this effect, and evidence is accumulating that Tregitopes (De Groot et al. Blood, 2008, 112:3303; natural T regulatory epitopes derived from IgG) provide beneficial immunomodulatory effects that parallel the effects of IVIG. In this presentation, we will provide evidence that Tregitope sequences derived from human IgG can reproduce immunomodulatory effects of IVIG in vitro and in vivo. In vitro, Tregitopes activate CD4+CD25+FoxP3+ natural regulatory T cells (nTreg). In vitro and in vivo, Tregitopes cause Tregs to produce IL-10, and to expand, and iTreg are induced. Induction and functions of nTregs have been examined in model systems such as D011.10 TCR transgenic mice, transplant of BM12 to C57BL/6, AAV-mediated gene transfer and EAE. Together, the data show that effector T cells, Th17, and Th9 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 that was comparable to, if not greater than IVIG. We will provide additional unpublished evidence demonstrating the antigen specificity of tolerance induction using Tregitopes in conjunction with target antigens, and discuss the relevance of Tregitopes to the treatment of human immune-mediated diseases.
Immune regulatory mechanisms, not alteration in Th1/Th2 skewing, are responsible for helminth-mediated protection against type 1 diabetes in NOD mice. (116.3)
Authors: Mitre Edward , Shi Yinghui , Torrero Marina , Mueller Ellen , Larson David , Soloviova Katerina , Stocker J. , Davies Stephen , Tarbell Kristin , Hubner Marc
Published: January 2023

Both Th2 immune responses and regulatory responses are increased during helminth infection and have the potential to be the mechanisms by which helminth infections protect against Th1-driven autoimmune diseases. We investigated these mechanisms by evaluating diabetes development in NOD mice infected with the filarial nematode Litomosoides sigmodontis in settings of depletion or absence of IL-4, regulatory T cells, IL-10, and TGFβ. Despite the absence of a Th2 immune shift, infection of IL-4-deficient NOD mice with L. sigmodontis prevented diabetes onset in all mice studied. Infections in immunocompetent and IL-4-deficient NOD mice were accompanied by increases in CD4+CD25+FoxP3+ regulatory T cell frequencies and numbers, respectively, increased proliferation of CD4+FoxP3+ cells, and increased production of TGFβ. Depletion of FoxP3+ T cells from splenocytes transferred into NOD.scid mice did not decrease helminth-mediated protection against diabetes onset. Continuous depletion of the anti-inflammatory cytokine TGFβ, but not blockade of IL-10 signaling, prevented the beneficial effect of helminth infection on diabetes. This study demonstrates that helminth-mediated protection against autoimmunity is not dependent on the induction of a Th2 shift but does require TGFβ. These findings suggest it may be possible to develop helminth-derived therapies for autoimmunity that induce protective regulatory responses without upregulating potentially harmful proallergic Th2 responses.
Diurnal variations in the phagocytic activity of zebrafish kidney leukocytes can be influenced by melatonin in a time-of-day dependent manner. (117.13)
Authors: Hannum Lynn , Ruksznis Catarina , Storer Alex , Wright Travis
Published: January 2023

Diurnal variations in immune system function result from rhythmic expression of clock genes. Their relationship with neuroendocrine regulators such as melatonin is not well understood. We explored the influence of melatonin on variations of phagocytic activity of zebrafish leukocytes. The percentage of kidney myeloid cells that phagocytosed pHrodo-labeled E. coli varied substantially throughout the day. The greatest increase in activity was between 6 AM and 10 AM, spanning the daily dark-to-light transition, a time when endogenous melatonin levels are falling to their daytime low. To determine whether extending the perceived night by prolonging the presence of melatonin would reduce the peak participation in phagocytosis observed at 10 AM, fish were exposed to exogenous melatonin for two hours beyond the beginning of the daily light period. Treatment with melatonin resulted in significantly lower participation in phagocytosis, similar to the normal 6 AM (dark phase) levels. The opposite effect was observed when fish received melatonin later in the day, prior to the evening light-to-dark transition. Fish exposed to melatonin prior to assay at 6 PM (late day) displayed a significant increase in phagocytosis over control fish, with participation levels surpassing normal 10 PM (early dark phase) levels. At both times of day, pre-treatment with the melatonin receptor antagonist luzindole prevented the changes in phagocytosis associated with extended melatonin presence.
Monocyte-derived tumor necrosis factor-α is essential for the protective immune response against Listeria monocytogenes (117.21)
Authors: Shi Chao , Williams Cassandra , Ling Lilan , Leiner Ingrid , Pamer Eric
Published: January 2023

Ly6Chi monocytes provide immune defense against infections. After being recruited from the bone marrow to sites of infection, Ly6Chi monocytes are localized to the bacterial foci and prevent infection from disseminating. In this study, we demonstrate that the antimicrobial functions of monocytes are largely dependent on their expression of TNFα, a pro-inflammatory cytokine. Specific ablation of TNFα expression from Ly6Chi monocytes significantly increases the susceptibility to L. monocytogenes infection in mice. Adoptively transferred monocytes lacking TNFα fail to provide protection, whereas their wild type counterparts do. TNFα produced by monocytes is required for organizing the cellular structure at hepatic lesions of infection that contains bacterial proliferation. It acts on monocytes and induces apoptosis in the cells harboring bacteria at the center of the lesion. Our findings provide insights for the function of TNFα and mechanisms by which monocytes contribute to host defense.
A predominance of PMN-II and M2b monocytes in alcoholics (117.9)
Authors: Kobayashi Makiko , Asai Akira , Tsuda Yasuhiro , Nakamura Kiwamu , Okita Yamato , Higuchi Kazuhide , Suzuki Fujio
Published: January 2023

Alcoholics are well-known to be immunocompromised hosts, and are susceptible to various infections. To determine the properties of effector cells in host antibacterial innate immunities, in the present study the functions and subsets of neutrophils and monocytes in peripheral blood of alcoholics were characterized. It has been reported that IL-12(+) neutrophils (PMN-I) and IL-12(+)IL-10(-) monocytes (M1 monocytes) are major effector cells in antibacterial innate immunities, while CCL2(+) neutrophils (PMN-II) and IL-12(-)IL-10(+) monocytes (M2 monocytes) are inhibitory on the generation of antibacterial effector cells. Neutrophils and CD14(+) monocytes, isolated from 10 alcoholics by Ficoll-Hypaque/dextran sedimentations and magnetic beads, were adjusted to 1 x 106 cells/ml and stimulated with or without 0.0075% SAC for 18 hrs (neutrophils) or 48 hrs (monocytes). Culture fluids harvested were assayed for cytokines by ELISA. In the presence of SAC, CCL2 was produced by neutrophils from alcoholics, while IL-12 was not detected in culture fluids of these neutrophils. Healthy donor neutrophils stimulated with SAC produced IL-12 (but not IL-10). Also, IL-10 and CCL1 were produced by monocytes from alcoholics, while healthy donor monocytes produced IL-12 under the stimulation. CCL17 and CXCL13 were not produced by monocytes derived from alcoholics. These results indicate that alcoholics are carriers of PMN-II and M2b monocytes.
Radiation combined with thermal injury: induction of a radio-resistant immature myeloid cell. (118.10)
Authors: Mendoza April , Brickey Willie , Neely Crystal , Ting Jenny , Cairns Bruce , Maile Robert
Published: January 2023

Increasing sophistication of terrorist attacks and recent nuclear energy crises have amplified the interest in the immunobiology of radiation combined with burn injury (RCI). Burn is associated with immunosuppression and increased mortality with lower doses of ionizing radiation. We hypothesize that RCI results in reduction of immune cell populations with a decrease in serum cytokine levels. C57BL/6 female mice underwent either a 20% TBSA full thickness contact burn or sham procedure. One burn group and one sham group received a single whole body dose of 5 Gy gamma radiation. Spleens and burn draining lymph nodes (dLN) were harvested 3 and 14 days later. Flow cytometry was performed to identify adaptive and innate cells. Serum was analyzed by multiplex bead array assay. IL-1β (day3), MCP-1 and IP-10 (days 3 and 14) were significantly elevated in RCI mice when compared to all other groups. At day 14, splenic and dLN T-cells, B-cells and neutrophils were significantly decreased after irradiation and RCI when compared to sham and burn alone. In contrast, numbers and percentages of Gr.1+CD11b+ cells were significantly increased. We confirmed these were not myeloid-derived suppressor cells as they did not suppress T cell proliferation in vitro. These data suggest that while RCI depletes immune cells, there are specific cytokine alterations and induction of a population of immature myeloid-derived cells which may act as a potential target for immune countermeasures.
Gene Expression of Defensin is Greatly Upregulated in Neonatal Neutrophils Following Labor (118.11)
Authors: Lawrence Shelley , Pereira Anne , Jiang Kaiyu , Frank Mark , Jarvis James
Published: January 2023

This investigation documents the variability of gene expression between neonatal and adult neutrophils at rest and following stimulation with lipopolysaccharide. Our hypothesis is that distinct gene expression differences will be observed between neonatal and adult neutrophils at rest and following stimulation with LPS. Methods: Cord (9) and adult (12) blood samples were collected per routine procedures. Neutrophils were isolated and divided into 3 groups, which were seeded at 1 X 10 6 ml into multiwall plates and incubated with or without LPS 10 ng/µL (Invivogen) for 0, 45, and 90 minutes. RNA was isolated and gene expression differences determined via the Illumina platform at the Oklahoma Medical Research Foundation. Signal intensities from each probe were quantitated, adjusted for local background, and quantile normalized (Matlabs; Natwick, MA) to adjust the marginal distribution of each sample. Summary: Neonatal cord neutrophils were up-regulated in > 75% of differentially expressed genes at each time point as compared to adults. This difference remained relatively unchanged prior to and following LPS stimulation. Furthermore, neonatal neutrophils demonstrated a 13-16 fold increase in the expression of defensins prior to and following LPS stimulation. Conclusion: Hierarchical clustering of gene expression results showed distinct differences between adult and cord neutrophils in unstimulated and stimulated states.
IMO-8400, a novel TLR7, TLR8 and TLR9 antagonist, inhibits disease development in lupus-prone NZBW/F1 mice (119.12)
Authors: Zhu Fu-Gang , Jiang Weiwen , Dong Yu , Kandimalla Ekambar , La Monica Nicola , Agrawal Sudhir
Published: January 2023

Several studies have implicated Toll-like receptors (TLRs) 7, 8 and 9 in the development and progression of lupus. We have developed IMO-8400, a synthetic DNA-based antagonist that has shown potent inhibition of TLR7-, TLR8-, and TLR9-mediated immune responses in cell-based assays and in non-human primates. To assess the therapeutic potency of IMO-8400, we have tested this compound in lupus-prone NZBW/F1 mice. Mice were injected subcutaneously with IMO-8400 at 1.9, 3.8 or 7.5 mg/kg once a week or 7.5 mg/kg once every two weeks from the age of week 21 to 33, and sacrificed at week 34. NZBW/F1 mice treated with IMO-8400 at the weekly dose of 3.8 or 7.5 mg/kg or 7.5 mg/kg every two weeks showed a significant reduction in urine protein and blood urea nitrogen levels in compression to that of untreated mice. Additionally, anti-DNA, anti-RNP and anti-SM IgG levels were also lower in treated mice than untreated controls. No treatment-related side-effects were observed. Histological examination of kidney sections revealed improvement in nephritis (reduced glomerular sclerosis and decreased interstitial inflammation), accompanied by decreased glomerular deposits of IgG immune complex in mice treated with IMO-8400. These results indicate that IMO-8400 suppressed autoimmune antibody production and improved renal function by inhibiting immune responses in lupus-prone NZBW/F1 mice. Thus, IMO-8400 has the potential for the treatment of lupus and other autoimmune diseases.
Neuroprotective effects of bee venom by suppression of neuroinflammatory responses in mouse model of Parkinson’s disease: role of CD4+CD25+Foxp3+ regulatory T cells. (119.2)
Authors: Chung Eun Sook , Kim Himchan , Bae Hyunsu
Published: January 2023

In the present study, we examined whether bee venom (BV) promotes the survival of nigrostriatal dopaminergic (DA) neurons in the MPTP-induced Parkinson’s disease (PD) mice model. Treatment of BV prevented degeneration of nigrostriatal DA neurons, increased striatal dopamine levels, and improved motor function. This nueroprotection afforded by bee venom was associated with the suppression of microglia and inhibition of recruitment of CD4+ T cells. Additionally, CD4+CD25+Foxp3+ Treg cells were significantly increased in vivo and in vitro after treatment of BV. Therefore, we carefully propose that neuroprotection of BV might be associated with its anti-inflammatory properties and could be employed as novel therapeutic agents for PD and other disorders associated with neuroinflammation.
Lymphotoxin reduces commensal diversity to enable diet induced obesity (120.2)
Authors: Upadhyay Vaibhav , Fu Yang-Xin
Published: January 2023

Commensal microbes exist in a state of symbiosis with host immunity and are essential for weight gain in diet induced obesity (DIO). Lymphotoxin α (LTα), a key molecule in mucosal defense, has been linked to obesity. We report that mice lacking LTα, LTβ, and LTβ-receptor (LTβR) resist DIO. The microbial communities of LTβR+/- mice and LTβR-/- mice differed on normal chow (NCD) and after high fat diet (HFD). The microbial community of LTβR+/- mice was less diverse after HFD, a hallmark of the “obese microbiome” in humans. On the contrary, LTβR-/- animals maintained similarly diverse communities to their NCD counterparts while on HFD. A specific microbe, Segmented Filamentous Bacteria (SFB), was cleared after HFD in LTβR+/- but not LTβR-/- mice and served as a marker of diversity loss. Transplantation of cecal contents from LTβR-/- animals conferred less weight gain than that of LTβR+/- mice to WT germ free mice, demonstrating a causal relationship between differing microbiota and weight gain. Housing LTβR-/- mice with their LTβR+/- siblings rescued weight gain, demonstrating horizontal transmissibility of the obese phenotype. HFD induced IL-23, an SFB associated cytokine, within the colon, but this induction was absent in LTβR-/- mice. Mice deficient in IL-23 (p19-/-) and the downstream cytokine IL-22 (RORγt-/-) also resisted DIO. Our data suggests that HFD induced inflammation reduces commensal diversity to enable weight gain, which may underlie the pathogenesis of obesity.
γ/δ T cells contribute to inflammation following long term low dose O3 exposure. (120.32)
Authors: Mathews Joel , Shore Stephanie
Published: January 2023

γ/δ T cells are involved in the responses to acute high dose ozone (O3) exposure (2 ppm for 3 hrs)( Matsubara, S et al, AJRCMB, 2009). However, elevated atmospheric O3 tends to persist not for hours, but for several days. The factors that determine pulmonary responses to short-duration, high-dose O3 exposure are different from those involved in responses to longer, lower-dose exposures and it is not know what role γ/δ T cells have in these longer term exposures. Consequently, we exposed mice deficient in γ/δ T cells (TCRδ-/-) and WT mice to O3 (0.3 ppm for 48 hrs). Bronchoalveolar lavage (BAL) was performed and the lungs were collected, digested, and analyzed by FACS. O3 caused an increase in numbers of γ/δ T cells, macrophages, and neutrophils in the lung tissue and/or BAL fluid. BAL and lung tissue neutrophils and macrophages were reduced in TCRδ-/- versus WT mice. BAL fluid IP-10, IL-6, MCP-1, LIF and KC were lower in TCRδ-/- versus WT mice. These chemokines are chemoattractive for macrophages and/or neutrophils and most can be produced by lung γ/δ T cells. In contrast, we found no difference in O3 induced lung damage between the WT and TCRδ-/- mice, as assessed by BAL protein or scores of terminal bronchiolar lesions on histological sections. Our data indicate that γ/δ T cells are involved in the initiation of inflammation following O3 exposure, potentially through the secretion of chemokines that recruit macrophages and neutrophils.
Skin-derived γδ T cells as a source of IL-17 in the lymph nodes and a possible initiator in contact hypersensitivity (121.22)
Authors: Nakamizo Satoshi , Egawa Gyohei , Miyachi Yoshiki , Kabashima Kenji
Published: January 2023

In the murine skin, there exist two subsets of γδ T cells, Vγ5+ dendritic epidermal T cells (DETCs) and Vγ4+ dermal γδ T cells. It remains unclarified about the role of skin γδ T cells in contact hypersensitivity and their homeostasis. Initially, to evaluate the mobility of cutaneous γδ T cells, we used kaede-transgenic mice which express photoconvertible fluorescence protein “kaede”. By exposing the skin to violet light, cutaneous cells changes its fluorescence from green to red. Using this labeling system, we demonstrated that γδ T cells migrated from the skin to draining lymph nodes (DLNs) even in the steady state and such transmigration was enhanced after hapten-application. Skin-derived γδ T cells were mainly Vγ4+ cells, suggesting that they were dermal origin. In addition, this subset produced a higher amount of IL-17 than either LN-resident γδ T cells or αβ T cells migrating from the skin. In addition, we found that γδ T cells assisted αβ T cells proliferation and production of IFN-γ and IL-17 in vitro. These data suggest that the main source of IL-17 in the DLN is γδ T cells from the dermis, and this population may play an important role to establish contact hypersensitivity response through modulating αβ T cell functions.
H1R signaling in antigen presenting cells is dispensable for eliciting pathogenic T cells in experimental allergic encephalomyelitis (123.14)
Authors: Saligrama Naresha , Noubade Rajkumar , Case Laure , Poynter Matthew , Teuscher Cory
Published: January 2023

The histamine H1 receptor (Hrh1/H1R) was identified as a shared autoimmune disease (SAID) gene in experimental allergic encephalomyelitis (EAE) and autoimmune orchitis, the principal AI models of multiple sclerosis (MS) and idiopathic male infertility, respectively. As a SAID gene, Hrh1/H1R can exert effects in multiple cell types including endothelial cells, T cells, and antigen presenting cells at critical check points during both the induction and effector phases of disease. In this regard, we showed that selective re-expression of H1R by endothelial cells in Hrh1-KO (H1RKO) mice significantly reduced disease severity whereas H1R expression by H1RKO T cells complemented EAE severity and cytokine responses. Given that the H1R has been reported to influence innate immune cell maturation, differentiation, chemotaxis, and cytokine production, which in turn influences CD4+ T cell effector responses, we selectively re-expressed H1R in CD11b+ myeloid cells of H1RKO mice to test the hypothesis that H1R signaling in these cells contributes to EAE susceptibility and/or T cell effector responses. We demonstrate that transgenic re-expression of H1R by H1RKO-CD11b+ cells neither complements EAE susceptibility nor T cell cytokine responses. These results further highlight the cell-specific effects that an AID gene can play in the pathogenesis of complex diseases such as EAE and MS, and the need for cell-specific targeting in optimizing therapeutic interventions based on such genes.
Type I IFN promotes IL-10 production from Th17 cells to suppress autoimmune inflammation (123.17)
Authors: Guo Beichu , Zhang Lixia
Published: January 2023

Recent studies reveal a considerable degree of heterogeneity and flexibility in T-cell lineage differentiation, particularly in the development of Th17 cells. However, how the immune system regulates the plasticity of CD4 T cells in vivo during pathogen infection or autoimmunity is poorly defined. In this study, we investigated the direct effects of type I IFN (IFNα/β) on Th17 cell differentiation and plasticity. In this study, we demonstrate that the plasticity of Th17 cells could be influenced by IFNα/β. Our data show that IFNβ treatment of T cells cultured under Th17 polarizing conditions resulted in reduced production of IL-17, but increased production of IL-10. Furthermore, type I IFN induced IL-10 production by antigen specific T cells derived from immunized mice. We also found that IFNβ treatment leads to shift from Th17 phenotype to Th1 phenotypes with upregulation of IFN-gamma in antigen-specific T cells in vivo. IFN-treated Th17 cells were able to inhibit EAE in vivo via adaptive transfer models. Together, our current findings demonstrate the direct effects of type I IFN on Th17 cells and reveal that Th cell plasticity is influenced by type I IFN during immune response.
B cell depletion - a double-edged sword (123.18)
Authors: Tai Ningwen , Wen Li
Published: January 2023

B cell depletion therapy has been proved beneficial in multiple T cell mediated autoimmune diseases, but the underlying mechanisms remain largely unknown. To investigate the immediate effect of B cell depletion on T cells in T1D, we used hCD20/NOD mouse model to determine the effect of short-term B cell depletion on the phenotype and function of effector T cells. B cells were depleted by a single dose injection of anti-hCD20 (2H7) in 7-8 week old mice. Mouse IgG was used as control. As expected, B cells in blood and secondary lymphoid organs were significantly reduced at day 3 and remained low until day 21. Surprisingly, splenic CD4+ and CD8+ T cells were also significantly reduced after B cell depletion. Several T cell subsets were dramatically reduced after B cell depletion, which included CD4-CD8-TCRβ+, B220+CD4+, CD40+CD4+ and CD45RB+CD4+ T cells. B cell depletion also induced ICOS expression in CD4+ T cells at day 3 and 7. Both CD4+ and CD8+ T cells expressed more TNF-α in 2H7-treated mice. T cell function post B cell depletion was tested in vivo using adoptive transfer approach. Either purified splenic T cells or FACS sorted CD40+CD4+ T cells from 2H7-treated mice (day 7) induced accelerated diabetes in NOD.scid or NOD Rag- mice compared with the same cells from control mice. Our results suggest that B cell depletion acts as a double-edged sword, which could enhance the function of T cell effectors to be pathogenically aggressive shortly after B cell depletion.
Lupus prone DBA/2 (DBA) CD4 T cells exhibit greater B cell help, poorer help for CD8 cytotoxic T lymphocytes (CTL) and induce more severe renal disease compared to B10.D2 CD4 T cells in parent-into F1 (P→F1) mice. (123.27)
Authors: Via Charles , Puliaiev Maksym , Soloviova Kateryna
Published: January 2023

Transfer of parental B6 T cells into B6D2F1 (B6→F1) mice results in acute graft-vs- host disease (GVHD) due to donor CD8 CTL that eliminate host cells. Donor CD8 CTL fail to mature in the opposite parental transfer (DBA→F1) resulting in chronic lupus-like GVHD and severe renal disease. To determine whether CTL failure and subsequent lupus like disease in DBA→F1 mice is an intrinsic CD8 defect or extrinsic due to poor CD4 help, female CD4 and CD8 T cells were purified from DBA or C57/Bl background, MHC identical (H-2d) B10.D2 donor T cells and transferred in all 4 possible combinations into female F1 mice. Transfer of B10.D2 CD4 +B10.D2 CD8 (D2+D2→F1) mice and DBA+DBA→F1 mice (positive controls) exhibited the expected acute and chronic GVHD phenotype respectively at 2 weeks. For mixed subsets, D2+ DBA→F1 exhibited a 2 week phenotype not significantly different from control acute GVHD indicating that normal (D2) CD4 help could correct a DBA CD8 CTL defect seen as absent KRLG-1 upregulation. By contrast, DBA+D2→F1 mice exhibited an intermediate phenotype significantly different from either control group indicating that normal D2 CD8 CTL maturation was significantly impaired with DBA CD4 help. Long term, only DBA+DBA→F1 and DBA+D2→F1 mice exhibited lupus autoantibodies or renal disease. Thus, DBA CD4 T cells are the primary determinant of clinical lupus in this model by virtue of greater help for autoreactive B cells and poorer help for down regulatory CD8 CTL.
Membrane-bound Fas ligand accelerates autoimmune disease in an induced murine model of systemic lupus erythematosus (123.3)
Authors: Bossaller Lukas , Rathinam Vijay , Schmidt-Lauber Christian , Moody Krishna , Bonegio Ramon , Fitzgerald Katherine , Latz Eicke , Marshak-Rothstein Ann
Published: January 2023

Lpr and gld, are loss of function mutations in the Fas death receptor and its ligand, respectively. However, in addition to its pro-apoptotic activity, FasL can also trigger the production of pro-inflammatory cytokines. The lpr and gld mutations normally result in a lymphoproliferative syndrome and exacerbate clinical disease in SLE autoimmune-prone strains of mice. Unexpectedly, the lpr and gld mutations were reported to attenuate disease progression in mice injected with pristane, an agent known to induce SLE. FasL is a transmembrane protein with a matrix metalloproteinase (MMP) cleavage site in the ectodomain. MMP cleavage inactivates membrane-bound FasL (mFasL) and releases a soluble form, sFasL, reported to have both antagonist and agonist activity. To better understand the impact of FasL cleavage, we have eliminated the cleavage site through targeted mutation, to produce the ΔCS line. ΔCS mice express more mFasL and fail to make sFasL. Pristane-injected ΔCS mice exhibit a markedly exacerbated disease profile associated with a striking change in ANA specificity, increased proteinuria and kidney pathology, a massive increase in spleen size due to infiltrating granulocytes and macrophages, a bias toward a Th2 response, and a trend towards increased numbers of Th17 T cells, compared to control mice. These results demonstrate that cleavage plays an important role in limiting the pro-inflammatory activity of FasL in the context of pristane-induced autoimmunity.
B7-H4 Ig treatment during relapsing-remitting EAE increases the number an function of regulatory T cells (123.37)
Authors: Podojil Joseph , Miller Steve
Published: January 2023

The present studies evaluate the potential therapeutic efficacy of B7-H4-Ig treatment in PLP139-151-induced relapsing-remitting EAE (R-EAE). The present data show that B7-H4 Ig inhibits CD4+ T cell proliferation and differentiation in the presence of various CD4+ effector T cell lineage-promoting conditions in vitro. We then go on to show that B7-H4 Ig treatment both directly and indirectly decreases effector CD4+ T cell activity by increasing regulatory T cell function and number. We also tested the ability of B7-H4 Ig treatment to reduce the severity of both PLP139-151-induced R-EAE in SJL mice and MOG35-55-induced C-EAE in C57BL/6 mice. Our results show that if B7-H4 Ig treatment is initiated on Day 0 of R-EAE, the primary disease relapse is inhibited as determined by mean clinical score and DTH responses to the spread epitope PLP178-191. Likewise, if B7-H4 Ig treatment is initiated during ongoing disease (~Day 20 post disease induction) in both R-EAE and C-EAE, disease severity and epitope spreading are decreased. Furthermore, this decrease in disease severity correlates with a decrease in the number of activated CD4+ T cells (CD4+CD44+) within the CNS and spleen, and a concurrent increase in number and functional suppressive capacity of regulatory T cells within the spleen (CD4+CD25+FoxP3+). Therefore, based on our in vitro and in vivo data, B7-H4 Ig demonstrates potential promise as a therapeutic in relapsing MS.
Role of the PD-1 pathway in regulating islet-reactive CD4 T cells during autoimmune diabetes (123.39)
Authors: Pauken Kristen , Fife Brian
Published: January 2023

Engagement of the inhibitory receptor PD-1 with its ligand PD-L1 is critical for preventing Type 1 diabetes in non-obese diabetic (NOD) mice. The mechanism by which PD-1 signaling limits self-reactive CD4 T cells is not well understood. To investigate this we transferred a low number of self-reactive BDC2.5 T cell receptor transgenic CD4+ T cells into prediabetic NOD mice. These cells became activated due to the presence of endogenous antigen. Following activation we administered anti-PD-L1 antibody to block PD-1 signaling and determined the affect on the BDC2.5 cells. PD-1 blockade resulted in an increase in BDC2.5 T cells in the spleen, pancreatic lymph node, and pancreas. This increase correlated with an increase in proliferation in the secondary lymphoid organs measured by CFSE dye dilution. However, it is unclear whether the increase in the pancreas was due to increased proliferation or enhanced recruitment. To investigate the requirement for PD-1 and/or PD-L1 on effector CD4+ T cells, we utilized genetically deficient BDC2.5 T cells. We determined that PD-1 on the BDC2.5 T cell was required to suppress proliferation and recruitment to the pancreas, whereas PD-L1 on the BDC2.5 T cell was dispensable. These data suggest that PD-1 is required on the autoreactive CD4+ T cells and under normal conditions will suppress proliferation in the secondary lymphoid organs and limit recruitment to the pancreas in a cell intrinsic manner.
IL-17-induced Rgs16 expression is essential for follicular T helper cell localization in the germinal centers of autoimmune BXD2 mice (123.44)
Authors: Ding Yanna , Hsu Hui-Chen , Druey Kirk , Wu Qi , Li Jun , Yang PingAr , Zajac Allan , Mountz John
Published: January 2023

Autoimmune BXD2 mice developed spontaneous germinal centers (GCs) and displayed increased number of CXCR5+ICOS+follicular T helper (Tfh) cells in the spleen. Although IL-21 but not IL-17 has been reported to be essential for the development of Tfh, there were significantly reduced PNA+Fas+GC B cells in the spleen of BXD2-Il21-/- and BXD2-Il17ra-/- mice. Interestingly, the frequency of Tfhs was 2.5-fold lower in BXD2-Il21-/- but 1.5-fold higher in BXD2-Il17ra-/- mice compared to wild type. Although increased, CXCR5+CD4 T cells were not specifically localized to the GCs in BXD2-Il17ra-/- spleens. Administration of AdIL-21 increased IL-17 levels in Tfhs and serum but had minimal effect on GC B cell frequencies in BXD2-Il17ra-/- spleens. In contrast, administration of AdIL-17 to BXD2-Il21-/- mice partially expanded GC B cells, suggesting that IL-17 acts downstream of IL-21 to facilitate the GC response. IL-21 stimulation of spleen CD4 T cells in vitro increased the frequency of Tfhs and upregulated expression of IL17R on Tfhs. IL-17 stimulated expression of Rgs16 and diminished CXCL13-mediated chemotaxis of CD4 T cells in vitro. Consistent with this, there were fewer GC CD4 T cells in BXD2-Rgs16-/- spleens in vivo. Our study suggests that IL-21 and IL-17 are required to promote the maximal autoreactive GC responses. IL-21 is important for Tfh development whereas IL-17 induced Rgs16 expression is required for Tfh localization in GCs to promote GC B-cell development.
The role of interleukin-21 in the pathogenesis of inflammatory bowel disease (123.45)
Authors: Poholek Catherine , Harrington Laurie
Published: January 2023

Th17 CD4 T cells are necessary for protection against pathogens but have also been cast as pathogenic in the context of many autoimmune diseases, including Inflammatory Bowel Disease (IBD). Th17 cells produce several inflammatory cytokines, including IL-17, IL-21, IL-22, and IL-26. Although these cytokines may act in concert to induce inflammation in colitis, IL-21 is a strong candidate for further scrutiny. IL-21 expression is increased in biopsies from patients with ulcerative colitis compared to healthy controls, and recent Genome Wide Association Studies have shown an association between the locus containing IL-2/IL-21 and IBD. We have shown that IL-21 signaling is required for the full induction of IBD in a murine model of disease. While others have shown in vitro that exogenous IL-21 acts to induce IL-17 production by CD4 T cells, our in vivo data suggests that IL-21 deficient cells are capable of producing IL-17A and IL-17F to a greater degree than IL-21 competent cells during IBD. In addition, our data disputes the previous finding that IL-21 suppresses the transcription factor Foxp3 as we have shown that IL-21 deficient T-regulatory cells express equal or less Foxp3 both in vitro and in vivo during IBD. Taken together, these data indicate that IL-21 plays a role in the induction of IBD that is separate from modulating the balance between effector and regulatory T cell responses. This work highlights a previously unrecognized role for IL-21 in the pathogenesis of IBD.
Optimizing conditions for the study of actively dividing T regulatory cells in flow cytometry (124.8)
Authors: Wilkinson Martha , Kwong Yu , Boyce Christopher , Elia Jeanne , Hingorani Ravi
Published: January 2023

The study of T regulatory (Treg) cells along with actively dividing cells can provide us with useful information in immunological studies since the cycling of Treg is different from the T effector cells. However, the buffer conditions may not always be compatible to combine FoxP3 staining with staining for dividing cells. We aimed to characterize the fixation and permeabilization buffers in the BD Biosciences catalog for the simultaneous staining of FoxP3 and Ki-67 in normal donors (both human and mouse). Six different buffer conditions were tested and experimental conditions optimized for detection of dividing Treg cells: BD Cytofix/Cytoperm™, BD Phosflow™ Perm I, BD Phosflow Perm II, BD Phosflow Perm III, BD Phosflow Perm IV, a new buffer system and ethanol. We have found that the new buffer system for fixation and permeabilization yields high quality data for FoxP3, Ki-67 and staining of surface markers. Other buffer systems also yield high quality data and can be used for the simultaneous detection of FoxP3 and Ki-67 with caveats that are discussed more in details within the poster.
Development of an in vitro pre-clinical cellular assay to predict induction of Cytokine Storm by therapeutic antibodies. (125.16)
Authors: Strizheva Galina , Randhawa April , Mulligan Jenny , O’Day Christine
Published: January 2023

Cytokine storm (Hypercytokinemia/Cytokine Release Syndrome) is an acute immune reaction consisting of a positive feedback loop between cytokines and immune cells, resulting in severe inflammation and organ failure. A clinical trial of immunotherapeutic anti-CD28 antibody TGN1412 in 2006 demonstrated the importance of thorough pre-clinical screening and need for more sensitive assays to predict cytokine storm. In that study, 6 healthy volunteers suffered from hypercytokinemia at doses 500-fold less than the lowest dose tested in non-human primates (NHP), showing the failure of standard pre-clinical safety approaches of that time. We have developed an in vitro assay to evaluate antibodies for induction of cytokine storm. Frozen PBMC from humans and NHP are cultured with test articles bound to the cell-culture plate and assayed for cytokine production and cell proliferation. Anti-CD28 superagonist antibodies, which have been shown to stimulate human PBMCs using similar mechanisms as TGN1412, are used as a positive control. Human PBMC produced significantly more IL-2, IL-6, IFN-gamma, TNF-alpha, MIP-1-alpha and several other cytokines compared to isotype control or untreated cells, while PBMC from NHP were non-responsive. The increased production of key pro-inflammatory cytokines in response to stimulation with anti-CD28 antibodies is accompanied by increased PBMC proliferation, while no proliferation increase is detected in isotype-treated or unstimulated human PBMC.
Host F4/80+ macrophages are increased after transplantation of embryonic stem cell derived hematopoietic progenitors (126.10)
Authors: Thompson Heather , McLelland Bryce , Manilay Jennifer
Published: January 2023

Mice transplanted with embryonic stem cell (ESC)-derived hematopoietic progenitors (ES-HPs) historically have displayed low levels of engraftment. We hypothesized that the innate immune system was an obstacle to ES-HP engraftment. To investigate this, we derived ES-HPs in vitro by growing D3-ESCs (strain 129, CD45.2, H-2b) on the bone marrow (BM) stromal cell line, OP9. We transplanted purified CD41+ ES-HPs into sublethally irradiated NOD.SCID.γc knockout (NSG) mice hosts (CD45.1, H-2g7) which lack B, T, and NK cells, but have functional macrophages. Host and donor-derived hematopoiesis was observed. However, at Day 18 post-transplant, ES-HP recipients displayed enlarged spleens that were primarily composed of host macrophages. The absolute numbers of host-derived splenic F4/80+ macrophages were increased 1.81 fold compared to untransplanted controls and 1.7 fold compared to BM transplanted controls. In vitro phagocytosis assays confirmed that NSG macrophages specifically engulfed allogeneic ES-HPs, but not allogeneic adult BM cells, supporting our in vivo results. Taken together, these results suggest that host F4/80+ macrophages may pose a barrier to allogeneic ES-HP engraftment. Importantly, these data suggest that adult macrophages preferentially phagocytose ES-HPs, which could reveal a novel mechanism of rejection unique to ESC-derived cells. Experiments to evaluate the effects of host macrophage depletion on ES-HP engraftment in vivo are ongoing.
Multiphoton intravital microscopy visualization of skin graft rejection in real time (126.14)
Authors: Kuligowski Michael , Loh Yik , Kwan Rain , de St Groth Barbara
Published: January 2023

The current paradigm explaining organ graft rejection postulates that rejection is initiated by graft resident dendritic cells that reach the draining lymph nodes via lymphatics and activate naive alloreactive T cells. These newly activated T cells then migrate into the graft. However up to half the T cells recognizing allogeneic MHC in adult animals are already be of memory phenotype, and thus capable of direct entry into a graft site without prior activation in LNs. This is not the result of previous exposure to allo-Ag, but stems from the fact that the allo-MHC-specific and Ag-specific T cell receptor repertoires overlap each other. These pre-existing allo-reactive T cells represent a major barrier to organ transplantation. To understand the contributions of memory cells to skin graft rejection, we are using intravital multiphoton microscopy to track cellular interactions between memory cells, DCs and lymphatic and vascular endothelium during graft rejection and acceptance. Preliminary studies suggest that DCs from grafts are unable to exit the surgical site, most likely as a result of surgical disruption of lymphatic drainage from the transplanted skin. Further work will investigate trafficking of graft-reactive CD4 effector and Treg cells into the graft site.
Macrophage Inflammatory Protien-2/CXCR2 Blockade Attenuates Acute Graft-versus-Host Disease while Preserving Graft-versus-Leukemia Activity (126.16)
Authors: Park Minhwa , Cho Kyung-Ah , Woo So-Youn , Ryu Kyung-Ha
Published: January 2023

Graft-versus-host disease (GVHD) is the principal complication after allogeneic bone marrow transplantation (BMT). Strategies that effectively separate the beneficial GVL effects from GVHD toxicity are necessary for enhanced survival after BMT. GVHD mice were induced by infusion of both donor BM cells and spleen cells into BU-CY conditioned recipient female BALB/c mice. Tumor-bearing GVHD mice were also made adding A20 cells. A total of 31 cytokines and chemokines were measured in the liver, intestine, and skin in GVHD mice by Luminex Multiplex assay. Four cytokines, G-CSF, KC, MIP-2, and IL-23, were commonly unregulated in target organs at the initial GVHD stage and were reduced by neutralizing MIP2/CXCR2 axis ligation. CXCR2 blockade on donor T cells and systemic administration of anti-MIP-2 neutralizing antibodies to BMT recipients caused a significant decrease in both systemic GVHD and target organ histopathology. GVL responses (BCL2 expression or B220+/CD19+ cells in BM and PB) after MIP-2/CXCR2 blockade were largely preserved and mortality was significantly delayed compared to control allogeneic BMT recipients. These data suggest that blocking the MIP-2/CXCR2 axis represents a novel strategy to separate the toxicity of GVHD from the beneficial GVL effects after allogeneic BMT.
Plasmacytoid dendritic cell enrichment of bone marrow allografts enhances GvL activity by facilitating donor T cell engraftment and Th1 polarization (126.25)
Authors: Darlak Katarzyna , Wang Ying , Waller Edmund
Published: January 2023

Allogeneic hematopoietic stem cell transplantation (HSCT) can cure patients with relapsed acute leukemia via a graft vs. leukemia (GvL) effect, but is complicated by graft vs. host disease (GvHD). Host dendritic cells (DC) following chemotherapy initiate GvHD in mouse models of allo-HSCT, but the role of donor DC is unclear. We have previously shown that transplanting purified plasmacytoid DC (pDC) along with HSC and T cells leads to Th1 donor T cell polarization and improved GvL activity. The goals of this study were: 1) To determine whether passive enrichment of pDC by depleting myeloid DC (mDC) from a bone marrow (BM) graft along with a fixed number of donor T cells would increase Th1 polarization and improve survival in leukemia-bearing mice, and 2) determine the role of IL-12 produced by donor pDC. Lethally irradiated B10.BR or FVB mice received BM and T cells from B6 or IL-12 KO B6 donors. Depletion of mDC from BM was achieved by FACS. Recipients of mDC-depleted BM had increased serum IFN-γ and increased donor T-cell proliferation and engraftment. Transplanting mDC-depleted BM enhanced survival without increasing GvHD in leukemia-bearing mice compared with undepleted BM. Enhanced proliferation and engraftment of donor T cells was not seen with mDC-depleted BM from IL-12 KO donors. These data show that pDC enrichment of BM allografts enhances donor T-cell Th1 polarization, engraftment, and GvL activity. The immune effects of mDC-depletion appear to be IL-12 dependent.
High affinity T cell receptor gives advantage to the homeostatic proliferation of naive CD8 T cells only under competitive pressure (126.28)
Authors: Ambros Tadeu , Zidan Abdel-Aziz , Pallin Maria , Montero Alberto , Lichtenheld Mathias , Díaz-Montero Marcela
Published: January 2023

The impact of T cell receptor (TCR) affinity during homeostatic proliferation has been demonstrated using naive CD8+ T cells with high and low affinity TCR derived from different precursors. In this study, we investigated the impact of TCR affinity using cells derived from the same precursor and selected under the same conditions, namely Pmel TCR transgenic (vβ13) naive CD8+ T cells, which respond to both high (hgp100) and low (mgp100) affinity peptides. Contrary to previous reports, we found that exposure of naive Pmel cells to either high or low-affinity peptide resulted in similar levels of homeostatic proliferation. However, in the presence of OT-1 cells and their cognate peptide (SINFKL), the levels of homeostatic proliferation of Pmel cells were higher after exposure to hgp100 than to mgp100 (1.85 vs 1.25 on day 8, 2.06 vs 1.20 on day 11, 2.57 vs 1.30 on day 15, 2.35 vs 0.89 on day 22). Interestingly, expression levels of memory markers (CD44lo to CD44hi, CD122lo to CD122hi and CD62Lhi to CD62Lhi) were similar under both circumstances. Overall, these results suggest that the difference seen in the effect of high-affinity TCR compared with low-affinity TCR on CD8+ cell expansion during homeostatic proliferation occurs only in the presence of competitive pressure. This difference may be balanced by a higher availability of low-affinity antigen, and thus may have important implications for vaccine design in the post-transplant setting.
The liver natural killer cells tolerate Alemtuzumab induction therapy in humans (126.3)
Authors: Ohira Masahiro , Nishida Seigo , Tekin Akin , Selvaggi Gennaro , Levi David , Tryphonopoulos Panaqiotis , Ruiz Phillip , Ricordi Camillo , Ohdan Hideki , Tzakis Andreas
Published: January 2023

Liver natural killer (NK) cells play a role in both graft acceptance and rejection in liver transplantation (LT). T cell depleting strategies have become an integral part of immunosuppressive regimens widely used for induction in transplant. Alemtuzumab (AL) is a recombinant humanized monoclonal antibody against human CD52. We observed early large granulocyte reconstitution in LT with AL. However, information is limited regarding the phenotype and function of NK cells in T cell depletion using AL. To investigate the role of NK cells in AL induction, we examine the phenotype and function of human NK cells derived from LT recipients and cadaveric donors. Absolute counts of T cells remained low for 6 months. In contrast, those of NK cells had emerged with larger number since early days after LT. Liver (n=7) contained high percentage of CD52-CD56+ NK cells (54.2 ± 9.0 % especially CD56 bright 81.2 ± 5.7 %), which suggested these NK cells could endure treatment of AL. It was statistically significant higher percentage than that of peripheral blood, spleen, and lymph nodes (25.1%, 28.4%, and 9.3 % respectively, P<0.05). Donor liver NK cells had stronger cytotoxicity after IL-2 stimulation in the presence of AL. These data indicated that NK cells in peripheral blood were depleted but NK cells in liver remained alive and active in LT patient with AL induction. These results implicate that liver NK cells play important roles in transplant recipients with AL induction.
Role of syndecan-1 in TRAIL-induced apoptosis in myeloma cells (127.18)
Authors: Wu Yung-Hsuan , Yang Chen-Ying , Chien Wen-Li , Lin Kuo-I , Lai Ming-Zong
Published: January 2023

Syndecan-1 is a transmembrane heparin sulfate proteoglycan which functions as co-receptor for cell signaling and extracellular matrix in many types of malignancies. TNF-related apoptosis inducing ligand (TRAIL) is an antitumor agent which initiates apoptosis through the assembly of death receptor-inducing signaling complex (DISC) after ligand binding. However, role of syndecan-1 in TRAIL -mediated cell death is unknown. Here, we elucidated the pro-survival role of syndecan-1 in multiple myeloma cell lines. Knockdown of syndecan-1 in H929 and RPMI8226 cells increased TRAIL-induced apoptosis. The expression level of signaling molecules downstream of TRAIL was normal in knockdown cells. Although the binding and internalization ability of TRAIL were still normal, an enhanced formation of DISC and caspase-8 activation were detected in knockdown cells. In addition, TRAIL-induced oligomerization of DR4 was enhanced in syndecan-1-knockdwon cells. Thus, syndecan-1 may regulate TRAIL-mediated cell death in the clustering of death receptors in multiple myeloma cells