bluuebunny/crossref_metadata_2025 huggingface dataset:
Monocyte phenotypes in systemic juvenile idiopathic arthritis (44.13)
Authors: Macaubas Claudia , Nguyen Khoa , Peck Ariana , Wong Elizabeth , Buckingham Julia , Goertz Yael , Deshpande Chetan , Alexander Heather , Chang Sheng-Yung , Sun Yue , Park Jane , Lee Tzielan , Begovich Ann , Mellins Elizabeth
Published: January 2023

Systemic juvenile idiopathic arthritis (SJIA) is a chronic autoinflammatory condition of childhood, characterized by remitting fever, transient rash, and relapsing arthritis. The association of macrophage activation syndrome with SJIA and clinical evidence implicating monocyte-derived cytokines IL-1 and IL-6 suggest a key role for monocyte in SJIA pathogenesis. We have previously found monocyte expansion during disease activity (flare), and a normal distribution of the monocyte subsets CD14hiCD16- and CD14loCD16+. We also observed elevated expression of monocyte surface markers CD14 and CD16 in the respective CD14hiCD16- and CD14loCD16+ monocyte subpopulations during disease flare and inactivity (quiescence), suggesting phenotypic alteration of monocytes in SJIA, independent of clinical status. We further analyzed phenotypic and functional alterations of SJIA monocytes in relation to disease activity and in comparison to age- and ethnicity-matched controls, specifically regarding development towards a M1- or M2- polarized phenotype. Gene expression profile, flow-cytometry based analysis of surface markers, baseline and LPS-induced production of cytokines and phosphorylation patterns in response to cytokine stimulation indicate that SJIA monocytes have a mixed M1/M2 phenotype during disease flare. Disease quiescence is associated not with normalization, but with an M2 phenotype, suggesting the existence of a state of compensated inflammation that is broke during disease flare.
An altered peptide of Chromogranin A is a potent antigen for the diabetogenic T cell clone BDC-2.5 (44.21)
Authors: Delong Thomas , Barbour Gene , Bradley Brenda , Reisdorph Nichole , Haskins Kathryn
Published: January 2023

Pathogenesis of type 1 diabetes is mediated by autoreactive T cells directed toward antigens in islet beta cells. We have recently identified Chromogranin A (ChgA) as the antigen target for three diabetogenic CD4+ T cell clones that were derived from the non-obese diabetic mouse, including the clone BDC-2.5. These clones respond to WE14, a natural cleavage product of ChgA, but only at very high peptide concentrations, suggesting that WE14 may not be the natural T cell ligand. Our question in this study was to determine whether a post-translational modification (PTM) could be involved in the formation of the natural ligand for the T cell clones. Although WE14 is expressed in beta cells, we were not able to identify the complete peptide sequence in purified antigenic fractions from beta cell tumors. Mass spectrometric analysis revealed the presence of an amino acid sequence corresponding only to the C-terminal region of WE14. Glutamine, which is in the N-terminal region of WE14, is a potential target for the enzyme transglutaminase. We treated the peptide WE14 with transglutaminase and demonstrated that this treatment significantly increased the antigenicity. Transglutaminases are known to deamidate or covalently crosslink proteins and we are currently investigating whether those modifications can be found in the natural antigen from beta cells. If transglutaminase is required for the generation of autoantigenic T cell epitopes, it could be an attractive therapeutic target.
Requirement of antigen cross-presentation for lupus tissue injuries: essential role of endosomal trafficking and proteosomal degradation (44.24)
Authors: Tsumiyama Ken , Takimoto Mai , Shiozawa Shunichi
Published: January 2023

Objective: Our ‘self-organized criticality theory’ explaining the cause of SLE shows that full-matured CTL generated via antigen cross-presentation induces lupus tissue injuries. Here we examine the molecular details of antigen cross-presentation to clarify how effector CTL is generated. Methods: Bone marrow-derived DC (BMDC) from BALB/c mice was cultured with fluorescent-labeled OVA. EEA1, calnexin and Sec61 were detected by using immunofluorescent staining. MG132 or primaquine (PQ) was repeatedly co-immunized with OVA to inhibit proteasomal degradation or endosomal trafficking to cell surface, respectively. Proteinuria and IFNγ-producing CD8+ T cell in spleen were examined. Results: In BMDC, OVA co-localized with EEA1, an endosomal marker. However, OVA was gradually separated from EEA1. In contrast, OVA never co-localized with calnexin, a ER marker. Sec61, a translocon, was co-localized with OVA, which indicated that OVA was transported from endosome to cytoplasm via Sec61. MG132, which inhibits proteasomal degradation, and PQ, which inhibits endosomal trafficking to cell surface, abrogated generation of CTL and development of nephritis. Conclusion: Endosomal trafficking, bypassing ER, is important not only in antigen cross-presentation but also development of lupus nephritis. Export of antigen from endosome to cytoplasm via Sec61 seems to be the first step in antigen cross-presentation, and subsequent the generation of lupus nephritis.
Lupus psychosis in IFNα transgenic mice (44.26)
Authors: Honda Eriko , Akiyama Chieri , Uchimura Chiaki , Hashiramoto Akira , Shiozawa Shunichi
Published: January 2023

Objective: We have shown that sole increase of interferon alpha (IFNα) in tetracycline-inducible transgenic (IFNα Tg) mice induces production of anti-dsDNA antibody and renal disease akin to human SLE. It has also been reported that IFNα is highly expressed in the brain and cerebrospinal fluids in patients with lupus psychosis. In this study, we have studied the contribution of IFNα to neuropsychiatric disease. Method: We made C57BL6/FVBN TetOp-mIFNα/EμSR-tTA transgenic female mice in which IFNα was expressed after cessation of doxycycline (Dox-off). Lupus psychosis was evaluated by behavioral test battery. Open field test (OFT), forced swim test (FST) and tail suspension test (TST) were performed in IFNα Tg mice 30 weeks after up-regulation of IFNα. Normal littermates (TetOp-mIFNα/EμSR-tTA: -/+) or IFNα Tg mice given doxycycline were used as control. Results: The locomotion score in OFT, which reflects exploratory activity, was lower in IFNα Tg mice than littermates (P<0.005). The activity counts in TST, which express depression and anxiety, were significantly lower in Dox-off IFNα Tg mice compared with Dox-on IFNα Tg mice (P=0.001). Increase of serum IFNα and up-regulated expression of IFNα in brain were confirmed by ELISA and immunohistochemistry, respectively. Further, we found that alopecia typical for SLE also developed at 24 weeks on up-regulation of IFNα in IFNα Tg mice. Conclusion: IFNα causes lupus psychosis and alopecia, important manifestations of SLE.
Impaired CD1d downregulation on B cells limits the cGVH response in Mertk-/- mice (44.28)
Authors: Shao Wen-Hai , Zhen Yuxuan , Finkelman Fred , Eisenberg Robert , Cohen Philip
Published: January 2023

The splenic marginal zone (MZ) contains specialized macrophages and B cells. Activation and migration of MZB into follicular (FO) regions of the spleen has been proposed as one of the mechanisms that regulate the development of autoreactive B cells. The Mer receptor tyrosine kinase (Mertk) mediates apoptotic cell clearance and regulates cytokine production. Although Mertk was previously not believed to be expressed on B cells, we recently reported unresponsiveness of Mertk-/- B cells to class II-alloactivation in a chronic GVH model. In the present study, we show that B cells from Mertk-/- mice but not WT B6 mice exhibited a decreased but more sustained calcium influx when stimulated by MHC-II cross-linking, indicating an intrinsic B-cell defect. In addition, in cGVH induced with CD4+ T cells from bm12 mice into Rag-/- mice reconstituted with mixed chimeras with bone marrows from Mertk-/- mice and C20 mice, autoantibodies came only from wild type (a allotype) and not from Mertk-deficient (b allotype) B cells. Compared to wild type, MZB cells from Mertk-/- mice were unable to down regulate surface CD1d expression and subsequent inclusion in the MZ, correlating with significant lower germinal center (GC) responses assessed by absolute GC numbers and PNA staining. Taken together, these data show that Mertk is needed for class II-activation of B cells, and that Mertk plays a role in CD1d down-regulation on MZ B cells, a potential mechanism limiting B-cell activation in cGVH.
Specific effects of Fli1 levels on T cells in the MRL/lpr lupus prone model (44.30)
Authors: Nowling Tamara , Morris Richard Erin , Harrell Maria , Basher Fahmin , Zhang John
Published: January 2023

The Ets factor Fli1 is implicated as a key modulator of lupus disease expression. Lowering the global levels of Fli1 in lupus mouse models or lowering the levels of Fli1 in hematopoietic cells in MRL/lpr lupus mice significantly improved kidney disease and prolonged survival. In this study, we analyzed the effects of reduced Fli1 levels on T cells in MRL/lpr lupus prone mice. Flow cytometry analyses of kidneys from MRL/lpr Fli1+/+ and Fli1+/- mice demonstrated a significant 35% decrease in total T cell numbers present in the kidney of 14 week old Fli1+/- mice, as well as significant changes in CD8+ (20% increase), CD4+ (40% decrease) and activated (30% decrease) T cells. We performed an adoptive transfer experiment with splenic T cells and B cells from 15-17 week old MRL/lpr Fli1+/+ and Fli1+/- mice. The cells were mixed in all combinations and transferred into Rag-1 deficient mice, which lack mature T and B cells. The Rag-1 deficient recipients receiving Fli1+/- T cells, regardless of the Fli1 levels in co-transferred B cells, expressed significantly lower levels of serum IgG and IgM. Mechanistically, the role of Fli1 in T cells has yet to be fully explored. However, these results suggest that Fli1 may play a role in T cell proliferation, apoptosis, or migration and/or T cell-B cell interactions. Importantly, the adoptive transfer results demonstrate that reducing Fli1 levels in T cells alone may have a positive therapeutic impact on the progression of lupus.
Activated natural killer cells enhance passive experimental autoimmune myasthenia gravis (44.35)
Authors: Wall Katherine , Saric Petar , Brown Paul
Published: January 2023

Myasthenia gravis (MG) is characterized by autoantibody-mediated loss of nicotinic acetylcholine receptors (AChR). However, antibody titers alone do not determine the severity of disease. Interleukin-12 (IL-12), a major inducer of interferon-gamma (IFN-γ) production, enhances experimental autoimmune MG (EAMG) in mice independently of the level of antibody and complement bound to AChR. IFN-γ is produced by various cell types, including CD8+ T cells, natural killer (NK) cells, and NK1.1+ T (NKT) cells. To investigate the roles of these cell types in disease enhancement, NK and NKT cells were depleted using anti-NK1.1 antibody prior to administration of IL-12 and passive transfer of monoclonal anti-AChR antibody D6 in B6 mice. For comparison, IL-12 treatment and passive transfer of mAb D6 was performed using TCRβ/TCRδ dual knock-out mice, which possess B cells and NK cells but lack functional T cells and NKT cells. IL-12 treatment worsened passive disease in wild-type B6 and TCR dual knock-out mice, but not in NK depleted B6 mice. Serum IFN-γ correlated with disease. These findings suggest that innate NK cells are the main source of disease-enhancing IFN-γ upon administration of IL-12. This provides a mechanism by which the innate immune response can enhance MG even when antibody levels remain unchanged.
Effects of Fli1 on T cell function in systemic lupus erythematosus (44.44)
Authors: Basher Fahmin , Harrell Maria , Richard Erin , Nowling Tamara
Published: January 2023

SLE, or lupus, is an autoimmune disease characterized by abnormal activation of T and B cells, resulting in autoantibody production and immune complex deposition in target tissues. T cells are one of the primary cell types infiltrating the kidney in lupus nephritis. Fli1 is a transcription factor implicated in the pathogenesis of lupus. Reduction of Fli1 expression resulted in decreased renal pathology and inflammation and increased survival in lupus-prone mice with specific effects on T cells observed. To determine the effects of Fli1 on T cell function, proliferation and apoptosis were measured in non-autoimmune prone (C57BL/6) and lupus prone (MRL/lpr) T cells stimulated with PMA and ionomycin. T cells isolated from C57BL/6 mice proliferated 170% more than MRL/lpr T cells regardless of Fli1 levels; however, no differences were observed in the rate of proliferation in Fli1+/- compared to Fli1+/+ T cells in either strain. Significantly more MRL/lpr T cells compared to C57BL/6 T cells, regardless of Fli1 levels, were undergoing apoptosis or dead. Interestingly, Fli1+/- T cells had a 26-35% increase in the percent of cells undergoing early apoptosis compared to Fli1+/+ cells in both strains. A chromatin immunoprecipitation array was scanned following IP with the Fli1 antibody of crosslinked T cell DNA. Highly enriched genes included IL-17F, IL-13, TNF, C3 and CEBPB. These results indicate that Fli1 may be involved in early apoptosis and inflammatory responses of T cells.
NKT cells in lupus prone NZB/W mice are abnormal and expression of Ly108 (SLAMF6) affects proinflammatory cytokine secretion and help for autoreactive B cells (44.46)
Authors: Tang Xiaobin , Strober Samuel
Published: January 2023

Systemic lupus erythematosus is an autoantibody mediated disease that causes immune complex glomerulonephritis as well as injury to multiple organs. Invariant natural killer T cells (iNKT) from non-autoimmune strains of mice regulate and suppress graft versus host disease, transplantation immunity and autoimmunity. We have shown that iNKT cells from lupus prone NZB/W mice are abnormal and promote rather than suppress spontaneous in vitro immunoglobulin and autoantibody secretion of autoreactive B cells. To determine the role of iNKT cells in the induction and augmentation of immune cell activation, we examined cytokine production of purified iNKT cells from NZB/W and control C57BL/6 mice in vitro. We found that upon stimulation of NKT stimulatory ligand, α-galactosylceramide, young NZB/W splenocytes secreted much higher IFNγ, IL-4 and IL-17 compared with C57BL/6 mice. With activation of CD3 and CD28, sorted NZB/W splenic iNKT cells, but not conventional T cells, also produced dramatically higher IFNγ and IL-4 than controls. Interestingly, we found the expression of Ly108, a SLAM family member (SLAMF6), affected cytokine production, and promoted help for immunoglobulin secretion by autoreactive B cells. Sorted NZB/W Ly108+ iNKT cells produced less IL-17 but more efficiently helped B cells secrete IgG but not IgM as compared to Ly108- iNKT cells. In conclusion, expression of the SLAM family member, Ly108, promoted the interaction of NZB/W iNKT cells with autoreactive B cells.
Structural and quantitative differences between membrane μ chains associated with surrogate or conventional light chains (45.1)
Authors: Hollander Nurit , Ben Moshe Natalie , Raviv Yasmin , Haimovich Joseph
Published: January 2023

Although it is well established that pre-BCR signaling governs proliferation and differentiation during B cell development, the components of the pre-BCR that are important for signaling are a matter of controversy. It has been suggested that signaling by the µ heavy chains (μHC) of the pre-BCR induces survival and differentiation, while the surrogate light chains (SLC) are essential for proliferation and clonal expansion. Using two variants of a murine B-cell line that differ only in surface expression of either BCR or pre-BCR, we demonstrated that surface µHC in the pre-BCR are of the high-mannose type only, while those in the BCR are of the complex type. We further demonstrated that unlike the BCR, the high mannose pre-BCR reaches the surface as a complex with calnexin. The amount of surface μHC in the pre-B cells is lower than that in B cells. We demonstrated that µHC in pre-B cells are in large excess compared to SLC. While LC in B cells are produced in enough amounts to assemble with all µHC, SLC are produced in insufficient amounts, leaving most µHC unassembled and therefore degraded. Igα and Igβ in pre-B cells are not a limiting factor as well, since they are produced in excess compared to SLC. Hence, the lower amount of the pre-BCR as compared to the BCR is probably due to insufficient amounts of SLC. In all, the structural and quantitative differences between μHC in the BCR and pre-BCR may account for their distinct signaling functions.
Dendritic cells express possible ligand(s) for CD38 (45.13)
Authors: Vences Catalan Felipe , Cedillo Barron Leticia , Santos Argumedo Leopoldo
Published: January 2023

CD38 is expressed in cells of the immune system, it has been implicated in several biological functions such as; chemotaxis, cell activation, cell proliferation, rescue or induction of apoptosis. It also has enzymatic activity converting NAD to ADPR and cADPR. It is not known how this molecule can perform different functions in different cells. It has been suggested that such biological functions could be mediated by different ligands, which would explain the pleiotropy attributed to CD38. CD31, present on endothelial cells, has been identified as a possible ligand for CD38; however, these results have not been confirmed in mouse. On the other hand, two molecules were identified in mouse dendritic cells as potential ligands but without further characterization. The aim of this work was to identify new possible ligands in a murine model using chimeric proteins of CD38. We designed three soluble chimeric proteins of the extracellular domain of CD38. One fused with CD8α, a second one fused with the Fc from the IgG1 immunoglobulin, both from human, and a third one containing only a V5 epitope and a histidine tag. The proteins were characterized by WB and enzymatic activity assays. All proteins formed soluble dimers and exhibited enzymatic activity. Using these proteins we were able to identify potential ligand(s) present on DCs but not on T and B lymphocytes from WT or CD38 KO immunized mice. This data suggest that DCs may play a role in CD38 function on B and T cells.
Prostaglandin E2 upregulates p53 mRNA and protein within activated human B lymphocytes (45.17)
Authors: Haque Shabirul , Lee Hyunjoo , Chiorazzi Nicholas , Mongini Patricia
Published: January 2023

Prostaglandin E2 (PGE2), a product of COX-2, plays a central role in inflammation and is elevated in pre-neoplastic tissue and malignancies, as well as in activated normal B lymphocytes. PGE2-induced signals via EP receptors modulate cAMP and/or calcium levels and can cross-talk with other signaling pathways (e.g. MAPK and NF-kB). p53 is a tumor suppressor with broad effects that include negative regulation of cell cycle, DNA repair, and induction of apoptosis. This study describes a role for PGE2 in regulating B cell p53 mRNA and protein. Purified human B2 cells from spleen/tonsil were pre-activated with stimuli resembling those found in sites of inflammation, i.e. surrogate C3dg-coated antigen + IL4 + BAFF. At d 4-5, cell density-limiting cultures were pulsed with PGE2 (50 nM) and harvested 1, 3, 8, 24, and 31 h later. p53 mRNA was monitored by both quantitative and qualitative RT-PCR. Exogenous PGE2 significantly enhanced p53 mRNA expression, in a time dependent fashion, over 3-31 h. This was accompanied by elevated mRNA for BID, a pro-apoptotic protein known to be transcriptionally regulated by p53, and linked to PGE2-elevated levels of p53 protein in replicating blasts. The above findings contrast with evidence that PGE2 promotes the expression of pro-survival Mcl-1 within replicating B cells. Taken together, they indicate that PGE2-mediated signals modulate both pro-survival and pro-apoptotic molecules in normal replicating human B lymphocytes.
The role of Erk pathway in the selection of autoreactive and non-autoreactive B cells (45.3)
Authors: Teodorovic Lenka , Rowland Sarah , Leahy Katelyn , Torres Raul , Pelanda Roberta
Published: January 2023

During development in the bone marrow, B cells undergo gene rearrangements, which lead to the expression of the B cell receptor (BCR) on the cell surface. Often times these BCRs have autoreactive specificities, requiring B cells to undergo tolerance mechanisms before undergoing further differentiation. Tonic BCR signaling, mediated by non-autoreactive BCRs, stops immunoglobulin (Ig) gene rearrangements, and allows cells to differentiate from immature into transitional and mature B cells. Immature B cells that receive antigen-mediated BCR signals are arrested in their differentiation and exhibit BCR downregulation and unresponsiveness to BAFF. Previous studies in our lab showed that the tonic BCR signal that drives immature B cell differentiation is mediated by the Ras-MEK-Erk pathway. However, we have not established whether activation of the Erk pathway is also involved in stopping Ig gene rearrangements. In mouse models where immature B cells co-express non-autoreactive (tonic signals) and autoreactive (antigen-mediated signal) BCRs, both tolerance and cell differentiation occur. Additionally, increased levels of pErk have been associated with positive selection of autoreactive B cells from the bone marrow. We hypothesize that tonic BCR signals overcome antigen-mediated signals by activating Ras-MEK-Erk pathway, leading to the inhibition of receptor editing and differentiation of autoreactive B cells. Results that test this hypothesis will be presented.
Intrathymic cortisone-resistant memory cells (46.11)
Authors: Vagida Murad , Pobezinsky Leonid , Pobezinskaya Elena , Kazansky Dmitry
Published: January 2023

It is known that central memory cells CD8+ are found in immunocompetent lymphoid organs, such as spleen and lymph nodes. Previously, we described the approach allowing to see and estimate selective proliferation of memory cells in MLR with heat-silenced (45 degrees, 1 h) allogeneic stimulator splenocytes. We immunized C57BL/6 (H-2b) mice intraperitoneally with mastocytoma P815 cells (H-2d). Two months later, we tested the proliferation of thymocytes in the MLR with heat-silenced syngeneic C57BL/6 (H-2b), allogeneic B10.D2 (R101) (KdIdDb) and third-party FVB (H-2q) splenocytes. Thymocytes of intact mice did not respond in MLR at all. Thymocytes of immune mice displayed small, but significant MLR responses to heat-silenced allogeneic splenocytes, but not syngeneic or third-party splenocytes. To test the hypothesis that thymic memory cells are cortisone-resistant, we treated immune mice with hydrocortisone. Injection of high dose of hydrocortisone (2,5 mg) 2 days before isolation of responder thymocytes resulted in highly elevated MLR responses. This response was antigen-specific because “immune” thymocytes responded only on heat-silenced R101, but not syngeneic or third-party splenocytes. As expected, in proliferating cultures, the ratio of CD4/CD8 was less than 1 due to proliferation of memory CD8+ cells, responding on H-2Kd MHC class I molecule represented on R101 thymocytes. Thus, we have found that the thymus contained memory-like T cells, which are cortisone resistant.
A role for viral dose in SLEC differentiation. (46.12)
Authors: Amoah Samuel , Alexander-Miller Martha
Published: January 2023

Our current understanding of effector cell differentiation suggests that inflammation plays an important role in driving an activated cell down the short lived effector cell (SLEC) versus memory precursor effector cell (MPEC) pathway. While IL-12 has been implicated as an important regulator of differentiation, this appears not to be the case for all infections, e.g. vaccinia virus (VV). To evaluate factors that may regulate effector cell differentiation under these circumstances, we investigated the effect of viral dose. We found that mice infected with a low dose of vaccinia virus had more B8R-specific cells at the peak of the response compared to those infected with a high dose of virus. Surprisingly, the observed increase in total number was the result of an increased SLEC population, leading to a response skewed toward SLEC. The increased SLEC in the low dose infected mice was not due to selective increases in proliferation or decreases in death (as measured by active caspase 3) in this population. In exploring this model we found that T regulatory cells were present at higher numbers in high dose infected mice. However, while depletion of Tregs led to an increase in the number of B8R specific cells, it did not result in a selective increase in SLEC. These data support a role for viral burden in effector cell differentiation, with decreased vaccinia virus dose driving the formation of SLEC.
A rapid method for the isolation of untouched naïve CD8 T cells from peripheral blood mononuclear cells (46.9)
Authors: McQueen Karina , Posarac Vesna , Fairhurst Maureen , Thomas Terry
Published: January 2023

Naïve CD8 T cells are, by definition, those that have been released by the thymus into circulation, but have not yet encountered their cognate antigens. Once they become activated they proliferate and generate effector T cells. We describe an easy and rapid method for the isolation of untouched naïve CD8 T cells from peripheral blood mononuclear cells (PBMC). Briefly, PBMCs are isolated by Ficoll-Paque PLUS density gradient sedimentation. Naïve CD8 T cells are then enriched using immunomagnetic, column-free, negative selection (EasySep™). Using EasySep™, unwanted cells are removed using antibody complexes recognizing non-naïve CD8 T cells and dextran-coated magnetic particles. Labeled cells are separated by placing the tube in an EasySep™ magnet. This procedure can be fully automated using RoboSep™. EasySep™ isolated naïve CD8 T cells are assessed by flow cytometry using a combination of antibodies specific for naïve, effector and memory cells. Naïve CD8 T cells are defined as CD8+CD45RA+CCR7+ and CD45RO-CD56-CD57-. Starting with PBMCs isolated from fresh blood and buffy coat, purities of 89±3% (n=8) are obtained (starting cell frequency 1.0-11.2%). EasySep™ isolated naïve CD8 T cells undergo robust proliferation as measured by flow cytometry of CFSE-labeled cells in culture with CD3/28 T cell expander beads. This simple method to isolate naïve CD8 T cells will be invaluable to researchers studying T cell differentiation, autoimmunity and infectious diseases.
The role of the activating versus inhibitory classes of G protein coupled histamine receptors on experimental allergic encephalomyelitis susceptibility. (47.2)
Authors: Saligrama Naresha , Noubade Rajkumar , Del Rio Roxana , Teuscher Cory
Published: January 2023

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) augmented by histamine (HA) and histamine receptor (HR) signaling. To help elucidate the mechanism behind HA and HR signaling on MS susceptibility, we utilize the experimental allergic encephalomyelitis (EAE) mouse model for MS and implement the use of traditional gene knockout studies. HA is known to exert its effects through four different G protein coupled receptors (GPCRs) designated as H1R, H2R, H3R and H4R. H1R and H2R couples to the Gαq/11 and Gs class of activating G proteins, respectively, whereas H3R and H4R couples to the Gi/o class of inhibitory G proteins. Each individual HR has been shown to influence EAE pathogenesis. Here we assessed the overall contribution to EAE susceptibility imposed by the two classes of GPCRs by generating C57BL6/J mice deficient for both H1R and H2R (H1H2RKO), as well as mice deficient for both H3R and H4R (H3H4RKO). The results of our study show that H1H2RKO mice exhibit decreased susceptibility to EAE, less severe neuroinflammation with a higher infiltration of T regulatory cells into the CNS, and decreased blood brain barrier permeability compared to H3H4RKO mice. Moreover, we show that splenocytes from immunized H1H2RKO mice produce significantly less IFN-γ and IL-17, which are cytokines known to exacerbate disease. Therefore, the activating class of GPCRs enhances EAE susceptibility compared to the inhibitory class of HRs.
Genetic loci on New Zealand black chromosome 1 lead to increased generation of T follicular helper and TH17 cells (47.23)
Authors: Talaei Nafiseh , Landolt-Marticorena Carolina , Noamani Babak , Pau Evelyn , Chang Nan , Wither Joan
Published: January 2023

Genetic loci on NZB c1 lead to antinuclear antibody (Ab) production and glomerulonephritis (GN). Using subcongenic lines, we found at least 5 genetic loci within a 35-106cM interval modulate disease. Notably, mice with a 70-100cM interval (c1(70-100cM)) develop high titre anti-dsDNA Ab, increased numbers of large germinal centers and severe GN a characteristic feature of lupus-prone mice with abnormalities of T follicular helper (TFH) and Th17 cells. In this study we determined whether variations in disease severity in c1 subcongenic mice are associated with differences in these subsets. The proportion of splenic TFH cells was significantly increased in c1(70-100) mice. Splenocytes from all c1 congenic mouse strains demonstrated increased production of IL-21 and IL-17 following anti-CD3/CD28 crosslinking which was greatest in c1(70-100) mice. The majority of IL-21 producing cells were TFH, many of which also secreted IFN-γ, but not IL-17. Immunization of 8-wk-old pre-autoimmune mice with ovalbumin (OVA) led to increased generation of TFH and Th17 cells in c1 (70-100) mice. This appeared to result in part from altered T cell function, as adoptively transferred OVA-specific TCR transgenic c1(70-100) T cells and naïve c1(70-100) T cells cultured in-vitro, demonstrated enhanced differentiation to TFH and IL-17-producing cells. Thus, genetic loci on NZB c1 promote differentiation of T cells to TFH and Th17 cells and this capacity correlates with the severity of disease.
Analysis of DRB1 sequence feature variant type associations with systemic sclerosis autoantibodies types and racial groups (47.28)
Authors: Marthandan Nishanth , Guidry Paula , Thomson Glenys , Arnett Frank , Karp David , Scheuermann Richard
Published: January 2023

A novel sequence feature variant type (SFVT) approach to genetic association analysis has been previously shown to identify parts of HLA proteins that are associated with the development of a variety of autoimmune diseases. Using the SFVT approach, we analyzed DRB1 typing data in a cohort of patients with Systemic Sclerosis (SSc) and demographically matched controls. We found no allele that was significant at p-value of 0.05 across all the three racial groups (African American, Caucasian and Hispanic) in the dataset. But, we found 23 SFVTs that were significant across the all three racial groups. In the analysis of SFVT association with the presence or absence of different auto-antibodies, we found a large overlap among the top significant sequence features (SFs). In spite of such overlap of SFs the top 100 SFVTs were exclusively associated with the presence of either anti-centromere or anti-topoisomerase but not both, indicating that different DRB1 sequences predispose to the development of different auto-antibody types. In addition, we found that SFVTs that differ by only a few amino acids can give rise to different autoantibodies in SSc patients (e.g. association of the antigen binding motif 67L_70Q_71R with anti-centromere, versus association of 67F_70D_71R with anti-topoisomerase). Tools for the conversion of four-digit HLA typing data into an SFVT data matrix and the calculation of chi-squared association statistics have been implemented in ImmPort (www.immport.org).
The P2Y11 receptor is associated with narcolepsy and with higher susceptibility to ATP induced cell death in T lymphocytes and NK cells. (47.5)
Authors: Kornum Birgitte , Lin Ling , Kassack Matthias , Kuipers Hedwich , Axtell Robert , Jennum Poul , Steinman Lawrence , Mignot Emmanuel
Published: January 2023

The sleep disorder narcolepsy with cataplexy is caused by the loss of hypocretin-neurons in the hypothalamus as the end result of what is hypothesized to be an autoimmune attack. Using a GWAS design, we recently published a significant association between narcolepsy and purinergic P2Y11 receptor polymorphisms. P2RY11 is a pseudogene in rodents and is thus poorly studied, although human data suggests involvement in extracellular ATP (eATP) induced immune cell death and chemotaxis. We found a P2RY11 genotype dependent effect on expression levels in PBMCs, with functional effects on sensitivity to eATP induced cell death. In further studies, we have found that a balance of P2Y11 and P2X7 expression may determine PBMC subtype sensitivity to eATP induced cell death. Notably, P2Y11 expression levels were CD56+>CD8+>CD19+>CD4+>CD14+, whereas P2X7 expression levels were CD14+>CD8+>CD4+>CD19+. In accordance with this, monocytes were more sensitive to eATP induced cell death than lymphocytes. We have also found that eATP-induced cell death is inhibited by P2X7 antagonists, increased by P2Y11 antagonists and decreased by P2Y11 agonists, in support of our hypothesis. Finally, we show that the genotype associated with narcolepsy affects mostly T lymphocytes and NK-cells. These findings suggest that a balance of P2Y11 and P2X7 activity may be important for selection of specific immune cells in the presence of eATP, and that this process may be important for narcolepsy pathophysiology.
CD30L null SJL mice exhibit reduced lymph node and spleen weights but support growth of transplantable SJL lymphoma RCS-X (48.29)
Authors: Tsiagbe Vincent , Podack Eckhard , Li Yu
Published: January 2023

The process by which endogenous retroviral superantigen (vSAg29) expressing SJL lymphomas (RCS) stimulate host CD4+Vβ16+ T cells, in order to elicit help (notably IL-4 and -5) for their growth, has been characterized as “reverse immune surveillance”. This response is facilitated by the high expression of an array of co-stimulating molecules on RCS cells, including B7.1, B7.2, 41BBL, CD40 and CD30. On the basis of their germinal center derivation, SJL lymphomas bear close resemblance to a subpopulation of human germinal center B cell lymphomas, such as CD30+ Hodgkin’s lymphomas. In our earlier studies, growth of transplantable SJL lymphoma (RCS-X) in vivo was inhibited by anti-CD30 mAb. While the evidence shows that this effect is indirectly on the lymphoma-responsive CD4 T cells, the mechanism of action was unclear. To shed light on the role of CD30-CD30L interactions in SJL lymphoma development, CD30L-/- were bred onto SJL background and tested for their support of transplantable lymphoma growth. A significant reduction in lymph node (17.4-54.7%) and spleen weights (21.5-38.2%) was observed for CD30 null mice, compared to wild type SJL mice. Despite this reduction in the lymphoid compartment, no reduction in lymphoma growth was observed. The results suggest that while CD30-CD30L interactions might be important for SJL lymphoma growth, other costimulating molecules might contribute significantly to the process of reverse immune surveillance in RCS lymphoma.
Tumor-associated macrophages suffocate NKT cells: a novel tumor escape mechanism and a target for therapy (48.7)
Authors: Liu Daofeng , Song Liping , Wei Jie , Gao Xiuhua , Marinova Ekaterina , Dotti Gianpietro , Metelitsa Leonid
Published: January 2023

Vα24-invariant Natural Killer T cells (NKTs) are important for antitumor immunity and immunotherapy. We have demonstrated that NKTs inhibit tumor growth indirectly via targeting tumor-associated monocytes/macrophages (TAMs) in neuroblastoma (NB), suggesting that tumor progression requires an escape from NKTs via yet unknown mechanism. Here, we demonstrate that NB cells via membrane-bound TNFα induce CCL20 production in monocytes and this effect is amplified in hypoxia. Anti-CCL20 neutralizing mAb strongly inhibited NKT-cell in vitro migration towards tumor-conditioned hypoxic monocytes and in vivo localization in NB/TAM xenografts in NOD/SCID mice. The immunofluorescent staining of primary NB tumors revealed selective CCL20 accumulation in TAMs. We also found that hypoxia impairs NKT-cell function, suggesting that NKT-cell trafficking toward CCL20-producing TAMs may serve as a hypoxic trap for tumor-infiltrating NKTs and other T cells. IL-15 protected NKTs from hypoxia in vitro and IL-15-transduced NKTs more efficiently inhibited tumor growth in metastatic NB model in humanized NOD/SCID/IL2rgamma(null) mice compared with parental NKTs. Thus, CCL20 production in hypoxic TAMs and consequent inhibition of NKTs reveals what we believe to be a novel mechanism of immune escape that can be reversed by adoptive immunotherapy with IL-15-transduced NKTs.
Pulmonary egress of CD8 T cells during influenza A virus infection. (49.10)
Authors: Jennrich Silke , Lee Michael , Dewberry Kristofer , Debes Gudrun
Published: January 2023

The capacity to migrate to and accumulate at infected sites is a requirement for antiviral effector functions of CD8 T cells. Memory/effector T cells efficiently enter sites of infection from the blood and subsequently leave the site via the afferent lymph. While T cell exit from extralymphoid tissue is regulated by the chemokine receptor CCR7 and alternative chemoattractant signals, it is controversial if effector T cells exit from the lung during acute respiratory infection. Here, we found that pulmonary T cells from influenza virus infected mice egress from the inflamed lung and migrate into draining lymph nodes. In addition, in vitro polarized (CD8+) Tc1 effector cells with the ability to protect against lethal influenza virus challenge were also able to egress from infected lungs upon intranasal transfer. Pulmonary egress of Tc1 cells was blocked by inhibition of chemoattractant signaling with pertussis toxin and significantly reduced by CCR7 deficiency. Our data demonstrate that effector T cell exit from the infected lungs is an active process, mediated by T cell expressed CCR7. We suggest that local T cell effector function and efficiency of host defense against influenza virus infection is controlled by the regulated expression of T cell exit receptors such as CCR7.
Route of priming regulates access to residual antigen and migration of memory T cells (49.12)
Authors: Jelley-Gibbs Dawn , Winfrey Carolyn , Woodland David
Published: January 2023

Memory T cells resident in peripheral tissues are key to establishing protective immunity to pathgens. There exist seemingly paradoxical concepts in the understanding of mechanisms regulating tissue-specific migration of CD8 memory T cells. One concept is that tissue-specific homing is imprinted by exposure to environmental factors during T cell priming. A contrasting concept is that memory T cells acquire tissue-specific homing properties through continued maturation in the periphery. Our mouse models of respiratory virus infection demonstrate that continued recruitment of CD8 T cells to the airways during steady-state memory is mediated by sequential events. Initial priming of CD8 T cells in the lung draining lymph node imprints memory CD8 T cells with the capacity to access residual antigen depots. Stimulation by residual antigen in the weeks to months following resolution of infection then promotes continued maturation and airway migration of CD8 T cells during steady-state memory. Interestingly, despite the formation of residual antigen depots following intraperitoneal, subcutaneous, or intramuscular infection, the resulting memory T cells ignore this maturation stimulus and fail to migrate to the airways despite exposure to lung inflammation. We hypothesize the site of priming is critical for imprinting memory T cells with the ability to access the additional signals required for continued maturation and effective migration of memory T cells to peripheral tissues.
Do immunostimulatory defective viral genomes arise during natural infections? (49.16)
Authors: Tapia Karla , López Carolina
Published: January 2023

The generation of defective viral genomes (DVGs) during the replication of viruses in vitro has been described for many different viruses including influenza, vesicular stomatitis and rabies. DVGs are truncated forms of the standard virus genome that are unable to replicate due to the lack of a functional viral polymerase. We have shown that DVGs have a potent ability to elicit the activation of dendritic cells in vitro demonstrating their exceptional capacity to trigger innate immunity. The presence of DVGs in viral preparations has been broadly correlated with protection against lethal doses of viruses. It was recently shown that RIG-I predominantly binds DVGs during infections with Sendai virus. The role of DVGs during natural infections is not known. Infection of bone marrow dendritic cells with different strains of Sendai virus showed that DVGs of different sizes are detected in all the viral preparations. In addition, infection with a Sendai virus strain that had been depleted of DVGs results in DVG generation at late points after infection correlating with the peak of viral replication. Interestingly, in mice infected with Sendai virus lacking DVGs we could also detect the appearance of DVGs at late times of infection. Together, these results show that DVGs can arise during the course of a natural virus infection stimulating exciting questions regarding their role stimulating anti-viral immune responses.
Cytolytic CD4 cells in the lungs of influenza-infected mice (49.17)
Authors: Marshall Nikki , Swain Susan
Published: January 2023

CD4 cells have been shown to acquire cytolytic activity (ThCTL) and demonstrate MHC class II-restricted killing of infected and transformed cells. We have identified a striking ThCTL subset in the lungs of influenza-infected mice that expresses high levels of Granzyme B, CD103 integrin, and CD107a and CD107b, markers of degranulation. This subset is not found in the spleen or lymph node. The ThCTL utilize a perforin-mediated mechanism to kill antigen-specific class II targets, and may play a role in killing influenza-infected airway epithelium which express high levels of MHC class II expression during influenza infection. ThCTL in the lung developed in mice deficient in Th1 polarizing cytokines IFN-γ and IL-12, further supporting their claim to being a unique subset of CD4 T cells. We are currently investigating the role of ThCTL in protection against influenza and the ability of ThCTL to develop into memory.
Apoptosis is necessary for functional T cell memory after influenza infection (49.19)
Authors: Thomas Paul , Dillon Christopher , Green Douglas , Sturm Oliver
Published: January 2023

Apoptosis can play a central role in the response to intracellular infections, both in the regulation of innate and adaptive immune responses and in the survival of the pathogen-infected cell. We investigated the effect of apoptosis in the response to influenza in various apoptosis deficient mouse models (Bim-/-, Bcl-2 and Akt tg) in vivo. Quantification of CD8 T-cells specific for immunodominant influenza epitopes (DbNP366, Db PA244, KbPB1703) revealed an increase in percentage and absolute CD8+ T cell numbers in the Akt tg, Bcl2 tg and Bim-/- primary, memory and secondary CD8+ T cell responses. The most striking effect was found in the Bim-/- model: a total ablation of the contraction phase after the CD8+ T cell response, leaving the number of flu specific memory CD8+ T cells abnormally high. Surprisingly, the immunodominance hierarchy in the memory Bim-/- response resembles that of a recall response (NP>>PA≈PB1). Their recall potential seemed blunted on a per cell basis compared to recall infections in wildtype mice, but after competitive adoptive transfer, the intrinsic recall division potential between the two memory cell populations was identical. However, Bim-/- CD8 T cells proved to be functionally deficient by cytokine production and effector activity, indicating a defect in the quality of memory. Thus, Bim-/- mediated contraction plays a role in selecting a memory precursor population with effector, rather than division, potential.
Development of Th17 and Treg cells during influenza viral infection in neonatal mice. (49.23)
Authors: Hoskins Samantha , Lines Louise , Garvy Beth
Published: January 2023

T cells in the neonatal lung are less activated than in adults and do not infiltrate the alveolar spaces in response to influenza virus infection. TGFβ is elevated in the lungs of neonatal mice, and since neonatal mice do not respond with a typical Th1 response, the Th17 and T regulatory (Treg) cells were evaluated. Neonatal mice had similar CD4 T cells expressing IL-17A compared to adults in the lung during viral clearance. In an adoptive transfer of adult and neonatal CD4 T cells into adult T cell receptor knockout mice (TCR-/-), neonatal T cells were able to enter the airways but failed to expand as robustly as adult T cells. In this model, neonatal CD4 T cells expressed more IL-17A than adult T cells and this coincided with increased numbers of neutrophils in the airway. Depletion studies will be used to examine the role of IL-17A and neutrophils in the clearance of influenza virus. In addition to a robust Th17 response, neonatal mice had higher levels of Tregs than adults, and these elevated levels persisted during influenza viral infection. Upon depletion of Tregs by anti-CD25 antibody, neonatal T cells still did not infiltrate into the alveolar spaces during an influenza viral infection. However, the anti-CD25 treated mice had a higher body weight and larger lymph nodes. To determine whether Tregs are responsible for the delayed viral clearance seen in neonatal mice, cytolytic T lymphocytes will be examined in neonatal mice without Tregs and plaque assays performed.
Rhinovirus has the unique ability to activate CD4 and CD8 T-cells, in the absence of Antigen-Presenting Cells (49.26)
Authors: Ilarraza Ramses , Wu Yingqi , Adamko Darryl
Published: January 2023

We hypothesize eosinophils (EOS) are key effector cells in asthma exacerbations caused by virus.We have shown that T-cells incubated with airway viruses activate eosinophil degranulation in vitro,after antigen-presentation (AP).Our recent data suggests Rhinovirus (RV) has the unique ability to activate T-cells directly.METHODS.Human blood monocyte-derived dendritic cells (moDC) were cultured with Respiratory Syncytial virus (RSV) or Rhinovirus (RV),washed and autologous T-cells and EOS were added.To inhibit AP,some moDC were treated with chloroquine during virus exposure.Also,T-cells alone (CD4+,CD8+,or mixed) were exposed to virus.Proliferation was measured by BrdU-ELISA and apoptosis by flowcytometry.EOS cysteinyl-leukotriene (Cys-LT) release was measured by ELISA.We also used agonists for TLR (2,3,4,7,8).RESULTS.RV induced Cys-LT release from EOS cultured with T-cells (n=6,p<0.001).Virus-moDC induced T-cell proliferation but,unlike RSV,RV was chloroquine-insensitive (n=5,p<0.001).T-cells alone showed proliferation (n=3-7,p<0.01) and apoptosis (n=4,p<0.001) with RV but not with UV-inactivated RV,or RSV.Proliferation was time-(n=3,p<0.01) and dose-(n=2) dependent.CD8 T-cells showed higher proliferation (n=2) and cell death (n=4,p<0.001) than CD4.TLR agonists alone did not induce T-cell proliferation.CONCLUSIONS.Results suggest RSV and RV induce T-cell activation differentially.Our data may help understand why RV is a potent inducer of asthma exacerbation in allergic patients