bluuebunny/crossref_metadata_2025 huggingface dataset:
DAP12 signaling in osteoclasts and macrophages is regulated by SHIP1 and PI3K binding to the ITAM of DAP12 (91.2)
Authors: Peng Qisheng , Humphrey Mary Beth
Published: January 2023

TREM2-DAP12 is critical for regulating TLR signals in macrophages and for normal osteoclastogenesis (OC). Negative regulation of DAP12 in OC and macrophages has not been reported and is the goal of this study. Our studies reveal that Src homology region 2(SH2)-containing inositol phosphatase (SHIP1) negative regulates DAP12 signaling. TREM2 antibody (Ab) cross-linking of mouse bone marrow macrophages (BMM) leads to association of SHIP1 with DAP12, not seen with control Ab. The SH2- domain of SHIP1 interacts with DAP12 in a phospho-tyrosine specific manner within the immunoreceptor tyrosine-based activation motif (ITAM). Signaling induced Pho-Akt, Erk1/2, VAV3 and Syk activation, and these signals were enhanced in SHIP1-/- BMM compared to wild type (WT) BMM. Cross-linking with anti-TREM2 monoclonal Ab, but not control Ab, lead to a 4 fold increase in OC generated from SHIP1-/- BMM compared to WT BMM. Analysis of the molecular mechanism reveals that SHIP1 regulates PI3K regulatory subunit P85a binding to the ITAM of DAP12. SHIP1-/- BMM displayed enhanced P85a binding to DAP12 in response to TREM2 cross-linking. Abrogation of PI3K activity leads to increased association of SHIP1 with DAP12. In OC and BMM, DAP12, SHIP1 and actin co-localize, and TREM2 stimulation leads to actin cytoskeletal changes. Together, these data reveal SHIP1 interacts with DAP12 and negatively regulates DAP12 through inhibition of PI3K binding to DAP12.
Differential PPAR γ and δ expression in F4/80hi and F4/80lo adipose tissue macrophages (92.8)
Authors: Bassaganya-Riera Josep , Guri Amir J , Hontecillas Raquel
Published: January 2023

Chronic inflammation and macrophage infiltration into adipose tissue are hallmarks of obesity. The objective of this study was to phenotypically and functionally characterize the subsets of adipose tissue macrophages (ATM). We had previously identified two distinct subsets of ATM based on surface expression of the glycoprotein F4/80. Changes in gene expression were examined on sorted F4/80lo and F4/80hi ATM by quantitative real-time RT-PCR and their phenotype assess by flow cytometry. While F4/80lo macrophages predominate in adipose tissue of lean mice, obesity resulted in increased accumulation of both F4/80lo and F4/80hi ATM. The F4/80hi population consistently expressed greater amounts of CD11c, MHC II, CD49b, and CX3CR1 on its cell surface and produced more TNF-α, MCP-1, and IL-10 than the F4/80lo subset, which contained a distinct Ly6ChiCCR2hi population. Gene expression analyses of the sorted populations showed that only the F4/80lo population produced IL-4, whereas the F4/80hi ATM expressed greater amounts of peroxisome proliferator-activated receptor (PPAR) γ, δ, CD36,TNF-α and toll-like receptor-4. Therefore, both the F4/80lo and F4/80hi ATMs are heterogeneous populations, with the F4/80hi population consisting of classically activated and deactivated macrophages expressing PPAR γ and δ and the F4/80lo population consisting mainly of IL-4-expressing alternatively activated M2 macrophages.
Lupus serum induces skin inflammation through TNFR1 pathway (93.1)
Authors: Deng Guo-Min , Liu Lena , Kyttaris Vasileios C , Tsokos George C
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by high levels of autoantibody and multi-organ tissue damage including kidney and skin. Skin manifestations are frequent in patients with SLE and because of limited understanding of the involved pathogenic mechanisms there is no specific treatment. Skin injury is also frequent in the lupus-prone MRL/lpr mice. We investigated whether lupus serum containing high levels of immunoglobulin can cause skin injury. We found that serum from patients and mice with lupus induced skin inflammation following intradermal injection in normal mice. Immunohistochemistry demonstrated that tumor necrosis factor receptor (TNFR) 1 but not TNFR2 was expressed in the skin lesions caused by injected lupus serum. The expression of NF-kB, iNOS and MCP-1 were also markedly increased in the induced skin lesions. The severity and incidence of lupus serum-induced skin inflammation were significantly decreased in TNF-alpha-deficient mice. Skin inflammation induced by lupus serum was abrogated in TNFR1-deficient mice. In contrast TNFR2 or IL-1R-deficient mice developed skin lesions comparable to those observed in normal mice injected with the same lupus sera. Our studies demonstrate that lupus serum causes skin injury and TNFR1 exerts a crucial role in the pathogenesis of lupus serum-induced skin injury by engaging TNFR1 but not TNFR2 or IL-1R. Therefore, disruption of TNFR1-mediated signaling process may be of therapeutic value in patients suffering of skin in SLE.
Enhanced effects of cigarette smoke extract (CSE) on inflammatory cytokine expression in IL-1beta-activated mast cells are regulated via the NF-kB pathway (93.16)
Authors: Chi David S , Lin Ta-Chang , Soike Thomas , Hall Kenton , Ha Tuanzhu , Li Chuanfu , Wu Zong Doa , Moorman Jonathan , Krishnaswamy Guha
Published: January 2023

Human mast cells are multifunctional cells capable of inflammatory responses and associated with allergy, asthma, and atherosclerosis. This study aims to examine effects and mechanisms of CSE on the expression of inflammatory cytokines in mast cells. Main- (Ms) and Side-stream (Ss) cigarette smoke were collected onto fiber filters and extracted in RPMI-1640 medium. Two ml of HMC-1 at 1 x 106 cells / mL were cultured with CSE in the presence or absence of IL-1β (10 ng / mL) for 24 hrs. The expression of IL-6 and IL-8 was assessed by ELISA and RT-PCR. NF-κB activation was measured by electrophoretic mobility shift assay (EMSA) and IκBα degradation by Western blot. Both Ms and Ss CSE significantly increased IL-6 and IL-8 production (p < 0.001) in IL-1β-activated HMC-1. CSE increased NF-κB activation and decreased cytoplasmic IκBα proteins in IL-1β-activated HMC-1. These results suggest that CSE enhance the expression of inflammatory cytokines through increased NF-κB activation and IκBα degradation, which may partially explain why cigarette smoke contributes to lung and cardiovascular diseases. (Supported by the Ruth R. Harris Endowment and RDC of ETSU)
Characterization of a selective Protein Kinase C theta (PKCθ) inhibitor for blocking T Cell responses (93.21)
Authors: Chaudhary Divya , Boschelli Diane , Bree Andrea , Brennan Agnes , Chen Joan , Deng Bijia , Fitz Lori , Goldman Samuel , Ho Melisa , Kasaian Marion , Lee Julie , Wang Daniel , Wei Shui-Qing , Williams Cara , Yang Xiaoke
Published: January 2023

PKC theta (PKCθ) regulates T cell activation in lung inflammation and airway hyperresponsiveness, and in mouse models of multiple sclerosis, arthritis, and inflammatory bowel disease. We investigated inhibition of PKCθ enzyme activity by a small molecule as a strategy for modulating T cell mediated responses. Screening and lead optimization identified Compound A, a potent PKCθ inhibitor in both enzymatic and cell based assays, with low nM inhibitory activities. Compound A was highly selective (>1000 fold) over 50 kinases including representative serine/threonine, tyrosine, and phosphoinositol kinases, and the conventional and atypical PKCs, PKCβ and PKCξ. Compound A was 10 fold selective against PKCε, and 60 to 100 fold selectivity against PKCδand PKCη, respectively. We found that compound A strongly inhibited IL-2 production in antiCD3/anti-CD28 activated whole blood, using both human and mouse blood, with submicromolar potency. Based on these findings, we evaluated compound A- mediated inhibition of anti-CD3 induced IL-2 production in vivo. The results show a dose dependent inhibitory effect of compound A in this 4 hour cytokine production model. Additional cytokines inhibited by compound A in this model are IFNγ, IL-4, and TNF. IL-5 production was not reduced. We propose that compound A is a selective PKCθ inhibitor for further evaluating blockade of T cell responses in inflammation.
The adenosine A1 receptor is a critical component in the modulation of cutaneous hypersensitivity by perceived stress (93.22)
Authors: Oliver Stephen J , Cronstein Bruce N
Published: January 2023

Differential effects of acute and chronic stress on skin immunity occur in the rodent restraint stress model, with acute stress enhancing and chronic stress suppressing cutaneous hypersensitivity (CHS). Extracellular adenosine levels modulate diverse biological activities in the CNS and peripheral tissues and serve an important protective function against physiological stressors such as inflammation and ischemia. We used the restraint stress model and the skin sensitizing hapten DNFB to test if perceived stress influences CHS through an adenosine A1 receptor-mediated mechanism. We subjected hapten-sensitized A1 receptor knockout (A1KO) mice and their wild type (WT) littermates to either acute (2.5h) or chronic (5h daily x 4 weeks) restraint stress, or no stress (controls), followed by hapten re-challenge of the pinna. In WT mice, 48h pinna swelling augmented with acute stress (p = 0.008) and suppressed after chronic stress (p = 0.038). In contrast, CHS responses in the A1KO mice were not affected by acute or chronic stress. Absence of the adenosine A1 receptor did not affect levels of plasma corticosterone or urine catecholamines under these stressful conditions but was associated with elimination of stress-induced neutrophila otherwise observed in WT animals. These results suggest that the adenosine A1 receptor pathway plays a critical role in how perceived psychological stress influences CHS responses.
Phosphoinositide 3-kinase p110delta regulates natural antibody production, marginal zone and B-1 B cell function, and autoantibody responses (93.23)
Authors: Durand Caylib A , Hartvigsen Karsten , Fogelstrand Linda , Kim Shin , Vanhaesebroeck Bart , Witztum Joseph L , Puri Kamal D , Gold Michael R
Published: January 2023

B-1 and marginal zone (MZ) B cells produce natural antibodies, make antibody responses to microbial pathogens, and contribute to autoimmunity. Although the delta isoform of the phosphoinositide 3-kinase p110 catalytic subunit (p110δ) is essential for development of these innate-like B cells, its role in mature B-1 and MZ B cells is not known. Using IC87114, a selective inhibitor of p110δ activity, we show that p110δ is important for B-1 and MZ B cell responses to anti-Ig antibodies, the Toll-like receptor (TLR) ligands LPS and CpG DNA, and the chemoattractants CXCL13 and sphingosine 1-phosphate. Moreover, we found that TLR-induced antibody responses by B-1 and MZ B cells, as well as the localization of MZ B cells in the spleen, depend on p110δ activity. Finally, we show that in vivo production of both protective and self-reactive natural antibodies requires p110δ and that p110δ inhibitors can reduce in vivo autoantibody responses. Thus targeting p110δ may be a novel approach for regulating innate-like B cells and treating antibody-mediated autoimmune diseases.

Supported by a grant to M.R.G. from the Canadian Institutes of Health Research, by NIH grants to J.L.W., and a grant to J.L.W. from the Leducq Foundation.
Glycogen Synthase Kinase-3β regulates antigen/SCF-mediated cytokine production and SCF-mediated chemotaxis in human mast cells (93.6)
Authors: Radinger Madeleine , Kuehn Hye Sun , Kim Mi-Sun , Metcalfe Dean D , Gilfillan Alasdair M
Published: January 2023

Glycogen Synthase Kinase-3β (GSK3β) is a serine threonine kinase downstream of PI3K, which has been implicated in cellular responses including tumorigenesis; cell growth, migration and survival; and cytokine generation. In this study we investigated the role of GSK3β in antigen/SCF-mediated mast cell responses. Short hairpin RNA (shRNA), introduced by lentiviral transfection, was used to knockdown GSK3β in human mast cells. Cytokine production and activation of transcription factors were measured by ELISA and immunoblot analysis. SCF-mediated mast cell chemotaxis was assessed with a transwell migration assay. GSK3β knockdown significantly decreased antigen/SCF-mediated GM-CSF and IL-8 production and SCF-mediated mast cell chemotaxis compared to control treated cells. The decrease in chemotaxis and cytokine production was associated with a pronounced reduction in antigen/SCF-mediated phosphorylation of JNK and the transcription factors c-jun and ATF2. These data are consistent with the conclusion that GSK3β is an essential component of the signalling cascade that regulates antigen/SCF-mediated transcriptional activity and chemotaxis in human mast cells. Thus, targeting GSK3β?may provide one means for down-regulation of these mast cell responses when associated with the allergic inflammatory reaction.

Supported by the Division of Intramural Research, NIH, USA.
High Fat Diet Induces Lung Remodeling in Apoe Deficient Mice: An Association with an Increase In Circulatory and Lung Inflammatory Factors (94.31)
Authors: Boulares Hamid A , Naura Amarjit S , Hans Chetan P , Zerfaoui Mourad , Errami Youssef , Ju Jihang , Kim Hogyoung , Kim Jong
Published: January 2023

Hypercholesterolemia is increasingly considered the basis for not only cardiovascular pathologies but also several complications affecting other organs including lungs. Here, we examined the effect of hypercholesterolemia on lung integrity using a mouse model (ApoE−/−) of high fat (HF) diet-induced atherosclerosis. A twelve-week HF diet regimen induced systemic production of TNF-α, IFN-γ, GMC-SF, RANTES, IL-1α, IL-2, and IL-12 with TNF-α as the predominant cytokine in ApoE−/− mice. Concomitantly, TNF-α, IFN-γ, and MIP-1α were detected in brochoalveolar lavage fluids of these mice, coinciding with lung inflammation consisting primarily of monocytes/macrophages. Such lung inflammation correlated with marked collagen deposition and an increase in matrix metalloproteinase-9 activity in ApoE−/− mice without mucus production or the mucus-promoting IL-13. Although TGF-β was undetectable in brochoalveolar lavage fluid of ApoE−/− mice on HF diet, it displayed a much wider tissue distribution compared to that of control animals. Direct intratracheal TNF-α-administration induced a lung inflammation pattern in wild-type mice that was strikingly similar to that induced by HF diet in ApoE−/− mice. TNF-α administration induced expression of several factors known to be critically involved in lung remodeling including MCP-1, IL-1β, TGF-β1, adhesion molecules, collagen type-1, and TNF-α itself in the lungs of treated mice. These results suggest that hypercholesterolemia may promote chronic inflammatory conditions in lungs that are conducive to lung remodeling potentially through TNF-α-mediated processes.
Differing contributions of CCR4, E-selectin and VLA-4 to the migration of CD4 memory and activated CD4+CD25+ T cells to dermal inflammation. (95.2)
Authors: Issekutz Thomas B , Gehad Ahmed , Al-Banna Nadia , Haidl Ian , Vaci Maria
Published: January 2023

CCR4 is on T cells in dermal inflammation and may mediate homing to skin. Our objective was to determine the expression and interaction of CCR4, E-selectin ligand (ESL) and a4β1 on memory and activated T cells in recruitment to dermal inflammation. A mAb to CCR4 (CR4.1) was developed. CCR4 was on ~10% of memory CD4 cells and 15% of these were ESL+. CCR4 and ESL were markedly increased on activated T cells. CCR4+ memory CD4 cells (memCCR4+) migrated 8-10 fold more to inflammation induced by cytokines, TLR agonists and DTH, than memCCR4- cells, and homed less to LNs. CCR4+ anti-TCR activated CD4 cells (actCCR4+) migrated only 50% better to skin than actCCR4- cells. E-selectin blockade inhibited 50-75% of actCCR4+, but not memCCR4 cell migration. a4β1 blockade had an inverse effect, i.e. inhibiting memCCR4+ more than actCCR4+ cells, while P-selectin blockade had no effect. Thus, CCR4 is on a subset of memCD4 cells with dermal tropism, but this selective homing is reduced on activated CD4+CD25+cells. The role of ESL and a4β1 also differs between activated and memory CCR4+ cells, with a decrease in the role of ESL and an increase in a4β1 with differentiation to long-term memory. (Supported by the CIHR).
TNFRSF25 promotes IL-17 and TL1A expression in murine CD4+ T cells (96.10)
Authors: Wolf Dietlinde , Deyev Vadim , Podack Eckhard R.
Published: January 2023

TNFRSF25 or Death Receptor 3 (TNFR25; DR3) is a member of the TNF-receptor family with TL1A (TNFSF15) as its ligand. TNFR25 is expressed constitutively on murine CD4+ and CD8+ T cells at low levels and up-regulated after T cell activation. IL-17 is a pleiotropic cytokine associated with inflammation and autoimmunity.

Using purified CD4+ T cells from transgenic mice over-expressing A. a full length (FL) TNFR25, B. a natural occurring splice variant, lacking exon 5 and 6 (Δ5,6), C. a dominant negative (DN) TNFR25, lacking the intracellular domain, and D. TL1A knockout mice, we investigated the role of TNFR25 signals in IL-17 expression. CD4+ T cells from full length and, to a lesser extend, those from Δ5,6 transgenic mice showed a significant increase in IL-17 secretion compared to wild type (wt) CD4+ T cells after primary and secondary stimulation under ThN and Th17 conditions in vitro. IL-17 levels in DN and TL1A-/- CD4+ T cells were comparable to wt. Cytokine levels of IFN-γ were comparable between wild type and transgenic/knockout mice, while Th2 cytokines were also increased in FL and Δ5,6 mice, as reported previously. Importantly, we detected highly elevated TL1A mRNA levels in un-stimulated full lenght CD4+ T cells.

Our data indicate that TNFR25 signals cause increased secretion of both Th2 and Th17 cytokines and elevate TL1A expression by CD4+ T cells. TNFR25 signals thus support inflammatory and allergic immune responses.
Cutaneous immune responses to 2, 4-dinitrochlorobenzene (DNCB): role of interleukin (IL)-2 (97.15)
Authors: Dearman Rebecca J , Portsmouth Craig , Cumberbatch Marie , Griffiths Christopher EM , Kimber Ian
Published: January 2023

Prolonged topical exposure of BALB/c strain mice to the contact allergen DNCB, or to the respiratory sensitiser trimellitic anhydride, results in T helper (h) 1 and Th2 cell polarisation, respectively. In addition, a single application of allergen results in discrete cutaneous cytokine profiles and the migration of Langerhans' cells (LC) with different tempos. Of note, DNCB only induced a rapid and transient increase in cutaneous IL-2. We have now explored the role of IL-2 in DNCB-induced LC migration. BALB/c mice received an intradermal injection of recombinant IL-2 or IL-1β (50ng/ear); the latter is known to provoke LC migration. LC densities were determined in epidermal sheets by immunofluorescence staining. Cytokine production by skin explants was measured by protein array. Intradermal injection of IL-1β decreased epidermal LC numbers significantly after 4 and 16h whereas IL-2 was without effect. Prior systemic administration of neutralising anti-IL-2 antibody failed to influence LC migration monitored 6h after DNCB treatment. Migration was blocked by treatment with an anti-tumor necrosis factor-α antibody, a cytokine regulator of LC migration. Administration of IL-2 did result in increased expression of cutaneous IL-17. These data show that IL-2 is not involved in LC migration, however, the selective up-regulation of IL-2 by DNCB may play a role in the subsequent polarisation of the immune response.
Anti-IL-21 monoclonal antibody reduces disease severity and inflammatory cytokines in a murine model of psoriasis and colitis (97.16)
Authors: Lewis Katherine E. , Bort Susan , Bilsborough Janine , Bontadelli Kristen , Maurer Mark , Waggie Kimberly , Kresja Cecile M. , Dillon Stacey R.
Published: January 2023

IL-21 plays a role in many autoimmune and inflammatory diseases, including psoriasis and inflammatory bowel disease (IBD). To investigate IL-21 antagonism in disease, we evaluated a neutralizing anti-mouse IL-21 mAb in a CD4+CD25- T cell transfer SCID mouse model of psoriasis and colitis. Mice treated prophylactically with IL-21 mAb had significant reductions in colitis and psoriatic-like skin symptoms compared to mice treated with PBS or isotype control mAb. IL-21 mAb-treated mice had 5-7-fold lower average lesional skin scores, 6-7-fold lower average clinical colitis scores and significant reductions in histological indices of colitis compared to controls. Mice treated with IL-21 mAb also had significantly lower serum IL-6, RANTES, TNF-α, and MIP-1β levels compared to controls, further supporting an anti-inflammatory role for IL-21 mAb. In a delayed dosing study, mice treated with IL-21 mAb starting 12 days after T cell transfer had significant reductions in colitis, with 3.5-4-fold lower average colitis scores than control-treated mice. Dose-related reductions in lesional skin scores were also observed. Efficacy of IL-21 mAb was equivalent to or better than an anti-mouse IL-12/23p40 active control mAb. These results demonstrate that IL-21 mAb reduces psoriasis-like disease and colitis in a murine model and suggest that an anti-human IL-21 mAb may have clinical utility in treatment of psoriasis and IBD.
Identification of the role of both IL-17RB and IL-17RA in mediating IL-25 induced activities and investigation of IL-17 and IL-25 in a mouse asthma and EAE model (97.9)
Authors: Rickel Erika , Siegel Lori , Rottman James , Hensley Kelly , Swart David , Tocker Joel E , Budelsky Alison
Published: January 2023

IL-25 is an IL-17 family ligand that promotes Th2 inflammatory responses. Intranasal (IN) administration of IL-25 into naïve mice induces pulmonary inflammation similar to allergic asthma. IL-25 has been reported to bind and signal through IL-17RB. Recently, it was shown that IL-17A signals through a heteromeric receptor composed of IL-17RA and IL-17RC. Using knockout (KO) mice and antagonistic antibodies, we investigated whether IL-25 also used a heteromeric receptor complex. Splenocytes from IL-17RA KO mice did not produce IL-5 or IL-13 in response to IL-25 and IL-17RA KO mice did not respond to IN administration of IL-25. Treatment of BALB/c mice with antagonistic antibodies to IL-25, IL-17RB or IL-17RA completely blocked IL-25 induced pulmonary inflammation and airway hyperresponsiveness. These data demonstrate that murine IL-25 mediated activities require murine IL-17RA. We also investigated the roles of IL-25, IL-17A, IL-17RB, and IL-17RA in a mouse asthma and EAE model. Blocking antibodies to IL-25, IL-17RA, or IL-17RB significantly attenuated the mouse asthma model, while anti-IL-17A had no effect. Conversely, blocking antibodies to IL-17RA and IL-17A significantly reduced disease in our EAE model, while anti-IL-25 had no significant effect. These data provide evidence that we can differentiate the activities of IL-25 and IL-17A acting through IL-17RA in different in vivo models of inflammation.

This research was supported by Amgen Inc.
CD38 modulates CXCL12-mediated chemotaxis: a novel mechanism in homing of chronic lymphocytic leukemia cells (98.7)
Authors: Deaglio Silvia , Vaisitti Tiziana , Aydin Semra , Bergui Luciana , Rossi Davide , Bonello Lisa , Chiorino Giovanna , Gaidano Gianluca , Malavasi Fabio
Published: January 2023

Human CD38, an ectoenzyme involved in the catabolism of NAD, can also bind the non-substrate ligand CD31, resulting in activation and proliferation of distinct lymphocyte populations.

CD38 is clinically used as a negative prognostic marker for chronic lymphcytic leukemia (CLL) patients. We adopted CLL as a model to study the relevance of CD38 in vivo. CD38 signals trigger proliferation and increase survival of CLL cells. Further, the simultaneous expression of CD38 and ZAP-70 defines a genetically distinct subset of CLL cells with heightened responses to CXCL12, a critical chemokine in CLL homing from blood to lymphoid organs.

Here we show that CD38 is directly involved in CXCL12-mediated homing. CD38 signals facilitate short- (ERK1/2 phosphorylation) and long-term (chemotaxis) effects induced by CXCL12. Assays using i) a specific inhibitor of CD38 enzymatic activities, ii) agonistic mAbs, and iii) microarray technology indicate that increased homing depends on the receptor functions of CD38. Conversely, blocking anti-CD38 mAbs significantly weaken CXCL12 responses. This is due to physical association on the membrane between CD38 and CXCR4 (the CXCL12 receptor), as shown by i) co-immunoprecipitation, ii) confocal microscopy and iii) measurement of CXCL12 binding to its receptor. These results indicate that CD38 modulates CXCL12 signals, representing a novel mechanism in CLL chemotaxis and homing.
Altered autoimmune response in NOD-Alox15null mice (99.13)
Authors: Morris Margaret A , Butcher M. , McDuffie M. , Nadler J.
Published: January 2023

12/15-lipoxygenase (12/15-LO) reacts with fatty acids to produce pro-inflammatory lipids, enhance IL-12 production by macrophages (MΦ), and its product 12-(S)-HETE induces pancreatic β-cell apoptosis at nM concentrations. Congenic NOD mice deficient in 12/15-LO (NOD-Alox15null) show a significant decrease in Type 1 diabetes (T1D) development (2.5 vs. >60% in ♀ by 30 wks). We tested the effects of the deficiency on the immune system in NOD-Alox15null vs. NOD mice. Adoptive transfer of diabetic (diab) or non-diabetic (non-diab) NOD, and non-diab NOD-Alox15null splenocytes (cells) determined ability of T1D disease transfer in NOD.scids. Both diab and non-diab NOD cells conferred T1D in NOD.scid hosts, but non-diab NOD-Alox15null cells did not (>8 wks post-transfer). NOD-Alox15null.scids were also injected with either NOD (diab and non-diab) or NOD-Alox15null (non-diab) cells. NOD cells still transferred disease, but NOD-Alox15null cells did not. As transferred splenocytes were mainly T and B cells, we tested for 12/15-LO mRNA expression in these subsets. 12/15-LO levels were minimally detectable, suggesting that immune effects are likely due to indirect effects on B and T cells. To study the mechanism, we looked at the role of 12/15-LO on APCs. Fewer MΦ infiltrate the pancreas of NOD-Alox15null mice at 4 wks of age compared to NOD controls. Also, MΦ from 10-wko NOD-Alox15null mice show reduced IL-12 expression compared to age-matched NODs. Therefore, 12/15-LO is a key regulator of the autoimmune response leading to T1D.
MOG intravenous tolerance modulates macrophage classical activation and antigen presentation in experimental autoimmune encephalomyelitis (EAE) (99.2)
Authors: li hongmei , Rostami Abdolmohamad
Published: January 2023

Macrophages act as the first line of self defense by mounting an inflammatory response to antigen exposure and as antigen presenting cells to initiate the adaptive immune response. Inhibition of macrophage activation is one of the possible approaches towards modulating inflammation. Intravenous (i.v.) tolerance has proved to be an effective method for ameliorating experimental autoimmune diseases. Whether macrophages are involved in tolerance induction is still largely undefined. In the present study we found that MOG i.v. down-regulated the expression of B7.1, B7.2 and MHC class II molecules, and reduced phagocytosis of GFP-transfected- Escherichia coli by both peritoneal macrophages and adherent splenocytes. Macrophages from tolerized mice were associated with a significantly impaired response of MOG-sensitized T cells to MOG. A distinct decrease in IFN-ã and IL-2 production by T cells in response to autostimulation was also observed. After MOG i.v. macrophages produced low levels of pro-inflammatory molecules IL-12, TNF-á, IL-1â, RANTES and MCP-1 and high levels of IL-10 and TGF-â. Administration of anti-TGF-â led to a reduction of IL-10 in MOG i.v-tolerized mice. These findings suggest that MOG i.v. inhibits macrophage classical activation and APC function and increases macrophage alternative activation, producing IL-10 and TGF-â, which, in turn, induce enhanced production of IL-10 in macrophages in MOG i.v. mice.
T lineage precursors from diabetes-prone NOD mice exhibit defective alpha-beta and enhanced gamma-delta T cell development (99.20)
Authors: Yui Mary A. , Rothenberg Ellen V. , Feng Ni
Published: January 2023

Because defects in various T lineages have been suggested to contribute to autoimmune diabetes in NOD mice, we sought to determine if early T cell precursors from NOD mice exhibit developmental abnormalities, which might affect later T cell lineage choices and responses. We previously reported that early T cells from NOD.Rag-/- mice undergo a spontaneous breakthrough at the first TCR-dependent checkpoint, a trait that appears to map near to two diabetes susceptibility genetic regions. We have also found that purified early T cell subsets from wild-type NOD mice undergoing differentiation in vitro exhibit profound defects in the generation and maintenance of DP populations, while gamma-delta T cell development is enhanced. NOD thymocytes also exhibit defects in development to the DP stage in vivo. These results show that NOD T cells have a lineage-specific abnormality at the first TCR-dependent checkpoint, which may select for DP cells with skewed signaling and/or survival characteristics, potentially altering positive and negative selection thresholds, T lineage choices, peripheral responses, and autoimmunity. (Supported by Juvenile Diabetes Foundation International and NIH R01AI064590 (to MAY).)
Identification of serum proteins that inhibit lupus immune complex stimulation of plasmacytoid dendritic cells (99.34)
Authors: Santer Deanna , Elkon Keith B
Published: January 2023

Regulation of IFN-α produced by plasmacytoid dendritic cells (pDC) is critically important in Systemic Lupus Erythematosus (SLE) as patients have an 'IFN-signature' which correlates with disease severity. We observed that, normalized for IgG, SLE patient cerebrospinal fluid (CSF) induced 800-fold more IFN-α by pDCs compared to paired serum due to potent inhibitory factors in serum.

Depletion of IgG from normal serum and addition of purified IgG to SLE immune complex (IC) stimulation assays revealed that normal IgG was one inhibitor in serum. Although the inhibitory capacity of IVIG has recently been attributed to a small subfraction of sialylated IgG in mouse models, sialylation of IgG did not significantly affect IVIG inhibition of IC stimulation of IFN-α. This result indicates either species differences or differences in pDC inflammatory pathways.

Deficiencies of early complement components predispose to SLE. Heat inactivation of normal serum and utilization of sera selectively deficient in early complement components revealed that a second serum inhibitor of IC mediated IFN-α stimulation was complement. Suppression of IFN-α produced by pDC was restored by adding back the missing complement component.

In summary, we have identified two serum factors that potently attenuate IFN-α induced by SLE ICs. Modulation of these components in vivo could be used therapeutically.
Role of IFN-γ in passive experimental autoimmune myasthenia gravis (99.4)
Authors: Brown Paul M , Wall Katherine A
Published: January 2023

In myasthenia gravis, anti-acetylcholine receptor (AChR) antibody is thought to cause damage through the activation of complement, via the classical complement pathway, in addition to causing internalization of AChRs. Previous research has shown that interleukin 12 (IL-12), the major inducer of IFN-γ production in the immune system, enhances clinical experimental myasthenia gravis (EAMG) in C57BL/6 (B6) mice. We performed passive transfer experiments with mAb399, a mouse anti-AChR monoclonal antibody recognizing the immunodominant mouse AChR epitope, using both B6 and IFN-γ-/- mice. The role of complement in disease induction in these mice was examined using fluorescent microscopy. When pre-treated with IL-12, B6 and IFN-γ-/- mice had similar amounts of antibody and complement bound at the neuromuscular junction, although IFN-γ-/- mice were resistant to EAMG development. This suggests that IL-12, through IFN-γ, may increase susceptibility to disease through mechanisms downstream of antibody and complement binding. (This work was supported by NIH AI 43061 and the OBOR Interdisciplinary Research Initiation Award)
Anti-apoptotic Genes are Upregulated in Tolerized CD8+ Regulatory T Cells Isolated from SLE-Prone Mice (99.41)
Authors: Skaggs Brian J , Ebling Fanny M , Singh Ram P , LaCava Antonio , Hahn Bevra H
Published: January 2023

Purpose: We studied the role of two different regulatory T cells (CD4+CD25+Foxp3+ Treg and TGFβ/IL10-secreting CD8+ Ts) in apoptosis pathways using a mouse model of systemic lupus erythematosus (SLE).

Methods: Ten-week old female NZB/NZW F1 mice were tolerized with a synthetic peptide based on anti-dsDNA VH sequences (pCons) that delays SLE symptoms or isotonic saline and killed one week later (n=6/group). After Treg and Ts isolation from spleens, gene transcript levels from three different pathways - apoptosis, T cell tolerance, and TGFβ - were examined by real-time PCR-based gene arrays (SA Biosciences). These platforms contain primers for 84 pathway-specific genes.

Results: Differences in Treg mRNA between the two groups were not apparent. In Ts, however, Bcl2, survivin, COX-2, Pak7, and IL-10 transcripts were significantly elevated more than two-fold in the pCons group. The proapoptotic gene Apaf1 was also significantly downregulated, implicating inhibition of specific caspases. Western blot confirmed altered protein regulation of these genes. Bcl-2, survivin and COX-2 are direct targets of the antiapoptotic transcription factor NF-κB, and Apaf-driven signaling pathways are involved in NF-κB regulation.

Conclusions: Results from these experiments provide additional insights into Ts apoptosis in SLE and could lead to novel therapeutic options designed to alter Ts biology.
IL-12/STAT4 SIGNALS ARE CRITICAL IN AUTOIMMUNE RESPONSE (99.44)
Authors: Yang Yu , Xu Jiangnan , Ochando Jordi C , Bromberg Jonathan S , Ding Yaozhong
Published: January 2023

Mice deficient in Th1 transcription factors T-bet or Stat4, are severely impaired in their ability to produce IFNγ and Th1 cells, and are resistant to the development of autoimmune diseases. Here, we introduced Stat6 deficiency into T-bet deficient mice to limit interference from elevated levels of IL-4/Stat6/GATA-3 Th2 polarizing signals, and to examine roles of T-bet and IL-12 in Th1 differentiation and colitis development. We found that eliminating IL-4/Stat6/GATA3 Th2 signals restored IL-12Rβ2 expression in T-bet deficient cells, and enabled T-bet deficient CD4 T cells to regain the ability to respond to IL-12 for IFNγ induction. Whereas T-bet deficiency abolished colitis development, transfer of Stat6 and T-bet double deficient CD4+CD25-CD45RBhigh T cells leaded to weight loss and colitis. Our data indicated that T-bet is not absolute for Th1 development and colitis induction, and demonstrated the critical role of IL-12/Stat4 in IFNγ production, Th1 development, and autoimmune disease development.
A self-peptide with large anchor residues binds IAd and induces Th2-like autoimmune gastritis (99.45)
Authors: Revilleza Maria Jamela Roxas , Levin Ditza , Mage Michael G , Teyton Luc , Robinson Howard , Shevach Ethan M , Natarajan Kannan
Published: January 2023

A mouse model of autoimmune gastritis with Th2-like phenotype in which BALB/c mice expressing a transgenic αβ TCR spontaneously develop the disease was developed in our laboratory. The TCR recognizes a 12-mer peptide from the alpha subunit of gastric parietal cell H/K ATPase presented in the context of IAd. The disease is characterized by slow and incomplete penetrance, anti-gastric mucosal cell antibodies, inflammatory eosinophilic infiltrates, parietal cell destruction and elevated Th2 cytokines. To investigate the contribution of MHC/peptide interactions to the initiation and maintenance of AIG, we generated a soluble recombinant version of the IAd/H/K ATPase889-900 complex for biophysical and crystallographic analyses. The 2.5 Å X-ray structure of the complex reveals rare features not seen in other IAd/peptide complexes: cysteine in P1, an imino acid in P6, and a large acidic residue in P9. This first structure of IAd in complex with an autoimmunogenic peptide reveals a previously unrecognized peptide binding motif for IAd with large anchor residues and with GLU in P9 encapsulated by a web of polar contacts that may contribute to stabilization of the complex and to maintenance of antigen presentation in disease.

This research was supported by the Intramural Research Program of the NIH, NIAID.
IL-15 prolongs CD154 expression via STAT5 engagement of the proximal CD154 promoter (99.48)
Authors: Cron Randy Q , Genin Anna , Orgun Nural N
Published: January 2023

CD154 expression is tightly regulated but increased expression on CD4 T cells in mouse models and humans with lupus contributes to disease. Since IL-15 is increased in lupus serum, we explored an IL-15 contribution to prolonged CD154 expression. Flow cytometry reveals increased CD154 levels on primary CD4 T cells following IL-15 plus PHA stimulation at later time points (48 hrs). Since IL-15 signals via STAT5 we explored the hCD154 promoter for possible STAT5 binding sequences (TTCNNNGAA). Gel shift assays with nuclear extracts from IL-15-activated primary human CD4 T cells reveal a proximal site (-65 bp) binds STAT5. Binding of STAT5 to the proximal CD154 promoter was confirmed in vivo by chromatin immunoprecipitation. Functionally, over-expression of wild-type STAT5 in primary CD4 T cells increases CD154 transcriptional activity in a dose-dependent fashion following T cell activation. Mutation of the STAT5 binding site eliminates this effect. Moreover, over-expression of dominant-negative STAT5 inhibits CD154 transcription. Similarly, STAT5 siRNA inhibits CD154 protein expression at late time points (48 hrs) post-T cell activation. Interestingly, STAT5 remains phosphorylated at late time points post-activation when IL-15R[alpha] expression is increased. The fact STAT5 binds the CD154 promoter in response to IL-15 provides novel approaches to CD154 inhibition via anti-IL-15 antibody, or by JAK3 inhibitors.
The role of Drak2 in the generation and function of regulatory T cells (99.54)
Authors: Hernandez Jeniffer B. , Walsh Craig M.
Published: January 2023

Autoimmune disease occurs via the escape of autoreactive T cells from central or peripheral tolerance mechanisms. T cells lacking the serine/threonine kinase Drak2 have a diminished activation threshold, suggesting that Drak2 serves as a quiescence factor to maintain tolerance to weak self-antigens. Drak2-/- mice are resistant to organ-specific autoimmune diseases such as EAE and IDDM. We have observed enhanced apoptosis in effector T cells derived from Drak2-/- mice, suggesting that Drak2 serves as a survival factor in effector T cells. Given the ability of Treg to prevent autoimmunity, we have investigated the potential that Drak2 may contribute to the generation or function of Treg. We have observed a significant increase in the ratio of Treg relative to conventional T cells (Tconv) in Drak2-/- mice. The increased Treg observed may be a consequence of enhanced differentiation of Drak2-/- Tconv toward adaptive Treg. Indeed, culture of naïve, Drak2-/- Tconv with plate-bound anti-CD3 alone resulted in the induction of a significant population of Treg. We hypothesize that the autoimmune resistance observed in Drak2-/- mice is due to the increased proportion of Treg and that DRAK2 may modulate regulatory T cell function. These results highlight the potential for inhibiting autoimmunity without affecting anti-pathogen responsiveness via DRAK2 blockade. This work was supported by NIH grant R01AI063419.
Immunosuppressive Ovarian Cancer-Infiltrating Dendritic Cells can be transformed into Immunostimulatory Cells through In situ CD40 and Toll-like Receptor 3 Stimulation (100.32)
Authors: Scarlett Uciane , Cubillos-Ruiz Juan , Nesbeth Yolanda , Martinez Diana , Fields Jennifer , Gewirtz Andrew , Ahonen Cory , Conejo-Garcia Jose
Published: January 2023

The individualized targeting of tumor-induced immunosuppression may contribute to the advancement of therapy against lethal epithelial tumors. We find that in a newly developed transgenic model of ovarian cancer, CD11c+MHCII+ dendritic cells are the most abundant leukocyte subset at tumor sites from early tumorigenesis. We also demonstrate (using a transplantable tumor model) that in the ovarian carcinoma microenvironment, these cells spontaneously engulf tumor materials and instead of promoting anti-tumor immunity, suppress T cell function. In situ co-stimulation of CD40 and Toll-like receptor (TLR) 3 on tumor-infiltrating dendritic cells reduced their L-arginase activity as well as enhanced their production of pro-inflammatory cytokines, augmented their capacity to process antigens, and up-regulated costimulatory molecules in vivo in mice and in vitro in human dissociated tumors. Correspondingly combined CD40/TLR agonists boosted measurable T-cell-mediated antitumor immunity and induced the rejection of otherwise lethal ovarian carcinomas. These results underscore the potential of transforming tumor-infiltrating dendritic cells from an immunosuppressive to an immunostimulatory cell type. Lastly, combined administration of synergistic CD40 and TLR3 agonists could enhance their individual therapeutic effects against ovarian and other lethal epithelial cancers.
Survivin: A Potential Target of Adoptive Immunotherapy for Pediatric Sarcomas (101.19)
Authors: Cui Yongzhi , Garber Haven , Zhang Hua , Mackall Crystal
Published: January 2023

Cell based immunity is driven by CD8+ cytotoxic T cells bearing TCR that recognize specific tumor-associated peptides bound to class I MHC molecules. Survivin, an inhibitor of apoptosis protein (IAP), is a tumor-associated antigen that is widely expressed in nearly all human cancer cells and undetectable in most normal tissues. Although there is evidence for cytotoxic T cell responses against survivin in patients, tolerance to self antigens can be a limiting factor in generating highly avid T cells capable of killing tumor cells. An alternate approach involves peptide immunization of HLA-A2 transgenic mice that express the human class I HLA-A2 molecule, which could in principle generate more highly avid TCRs to human antigens. In this study, we vaccinated A2 transgenic mice with a modified survivin epitope, Surv96M (LMLGEFLKL) and successfully generated CD8+ CTL clones that react against Survivin96-104 and a human pancreatic cancer line. The α- and β- chains for the TCR of reactive CTLs were cloned and inserted into an retrovirus vector incorporating 2A linker peptides between coding sequences of α- and β- chains of TCRs against survivin. Transduction of survivin specific TCR retrovirus constructs into human PBMCs yielded up to 70% TCR redirected lymphocytes and specific effector function as measured by IFNgamma release following co-culture with peptide-pulsed targets and one A2+ pediatric osteosarcoma line. Ongoing work is underway to increase the TCR affinity to potency.
Targeting of breast tumors by an HLA A*0201/MIF specific antibody (101.23)
Authors: Hawkins Oriana , Verma Bhavna , Neethling Francisca , Caseltine Shannon , Lightfoot Stanley , Blakey Gregory , Dooley William , Weidanz Jon , Hildebrand William
Published: January 2023

We previously utilized secreted HLA technology to identify a Macrophage Migration Inhibitory Factor (MIF19-27) encoded peptide as unique to the HLA-A*0201 molecules of breast cancer derived cell lines. The aims of this study were to (1) generate a mAb specific for the A*0201/MIF complex, (2) to assess A*0201/MIF expression on primary tumors, and (3) to evaluate the cytotoxicity of the antibody to breast cancer cell lines and tumor xenografts. The RL21A mAb specific for A*0201/MIF was generated by immunizing mice with peptide/MHC complexes. Matched tumor and normal cryopreserved breast tissues were HLA typed and A*0201 tissue sections were stained with RL21A. Staining of the normal and primary tumors were compared for HLA A*0201 expression and for A*0201/MIF staining. The RL21A TCRm antibody specifically stained HLA-A*0201 positive breast tumors. This confirms the presentation of the MIF on primary breast cancer tissues and validates cancer epitope discovery using secreted HLA technology and cancer cell lines. RL21A was capable of inducing potent complement mediated (CDC) and cell mediated cytolysis (ADCC) as measured by in vitro lactate dehydrogenase (LDH) release. In addition, RL21A reduced tumor burden in a prophylactic tumor model, employing an aggressive BT20-A2 xenograft. Here we demonstrate that the HLA-based discovery of cancer epitopes can lead to the generation of mAb that specifically recognize and destroy tumors via the A*0201/peptide complex.
Origin and number of viral peptides sampled by class I MHC following West Nile Virus infection (130.27)
Authors: Laughlin Amanda , McMurtrey Curtis , Bardet Wilfried , Fogle Matthew , Wahl Angela , VanGundy Rodney , Buchli Rico , Hildebrand William
Published: January 2023

West Nile Virus (WNV) was introduced into the United States in 1999 and it has emerged as the most common cause of arboviral neuroinvasive disease in the U.S. An estimated 1.65 million infections occurred between 1999 and 2008 and there is no specific treatment or vaccine available. WNV-infected cells can be detected and destroyed by the CD8+ cytotoxic lymphocytes via the presentation of viral peptides by major histocompatability complex class I molecules. Currently, no one knows how many WNV derived peptides decorate the MHC of infected cells, nor do we know which viral proteins MHC molecules sample. Understanding the number and origin of viral peptides available for immune recognition is a key prerequisite in the development of antiviral immunotherapeutics. Using an HLA-A and an HLA-B molecule, we previously reported that a given class I MHC molecule samples 2-6 viral peptides. Here we test this observation with additional HLA-A and HLA-B by secreting A*0101 and B*2705 from uninfected cells and then from WNV infected cells. Peptides eluted from the infected and uninfected class I molecules were compared by mass spectrometry, and peptides unique to infected cells were sequenced by tandem mass spectrometry. The resulting data demonstrate that different class I MHC molecules consistently present a like number of WNV peptides for immune recognition and that the viral peptides originate from clusters or “hot spots” within the viral polyprotein.
DIRECT ORCHESTRATION OF CD8+ T-KILLERS BY CD4+ T-HELPERS AND CD+ T-LICENSED ANTIGEN PRESENTING CELLS (130.33)
Authors: Xiang Jim
Published: January 2023

We previously proposed a CD4+ Th-APC concept by demonstrating that in vitro ovalbumin (OVA)-pulsed dendritic cell (DCova)-activated CD4+ T cells acquired peptide/major histocompatibility complex (pMHC) I from DCova and are capable of stimulating CD8+ cytotoxic T lymphocytes (CTLs). In this study, we developed a novel immunization protocol to “dissect apart” respective roles of DCova, CD4+ and CD8+ T cells separately in an in vivo environment with close to physiological condition. We first used anti-CD4 and anti-CD8 antibody (Ab) and deptheria toxin (DT) for in vivo depletion of CD4+ and CD8+ T cells and DCova derived from DT receptor (DTR)-CD11c transgenic mice, respectively, at different time points of the protocol. We then assessed the priming of CD8+ CTL responses in blood using FITC-CD8 and PE-Kb/OVA257-264 tetramer staining by flow cytometry. We found that (i) mice that have CD4+ T-licensed DCova but lack CD4+ T cells by anti-CD4 Ab treatment during CD8+ CTL priming, induced CTL effector/memory responses, (ii) CD4+ T cells with in vivo acquired pMHC I, but not those without pMHC I acquisition stimulated CTL responses, and (iii) APC-primed CD4+ T cells mounted more efficient CTL effector/memory responses than CD4+ T-licensed DCova. Our results thus provide a clear in vivo evidence for a direct orchestration of CD8+ T-killers by CD4+ T-helpers and CD4+ T-licensed APCs. This conceptual advance may have great impacts in re-evaluation of antitumor and autoimmune responses.