bluuebunny/crossref_metadata_2025 huggingface dataset:
Self antigens are partial agonists for NKT cells (B221)
Authors: Wang Xiaohua , Besra Gurdyal S , Gumperz Jenny E
Published: January 2023

NKT cells are regulatory T cells that recognize glycolipid antigens presented by CD1d. The antigens recognized by NKT cells include self and foreign lipids, but it is unclear whether these elicit different functional responses. Here we show that presentation of self antigens to human NKT cell clones stimulates quantitatively and qualitatively different responses than the potent exogenous antigen α-GalCer. Self antigen recognition only efficiently activated secretion of GM-CSF and IL-13, whereas α-GalCer stimulated full cytokine production (GM-CSF, IL-13, IL-4, IFNγ, and IL-2) as well as cytotoxicity and proliferation. Recognition of self antigens resulted in TCR down-regulation, CD69 up-regulation, and ERK phosphorylation, indicating that TCR-mediated signaling had occurred. However, no NKT cell Ca++ flux was detected in response to self antigen, although α-GalCer stimulated robust Ca++ signaling. Recognition of self antigens in the presence of ionomycin, which artificially induces Ca++ flux, resulted in full functional responses, including production of IL-2 and proliferation. Conversely, stimulation by α-GalCer in the presence of cyclosporin A to block calcium signaling, preferentially inhibited IL-2 secretion. Hence, NKT cell recognition of self antigens is associated with a defect in calcium signaling, and results in a partial activation phenotype.
Tetramer staining of Anaplasma marginale specific CD4 T cells following major surface protein immunization reveals the rapid loss of specific T cells in response to infection (B23)
Authors: Brown Wendy C , Han Sushan , Mwangi Waithaka , Palmer Guy H , Norimine Junzo
Published: January 2023

Immunization with A. marginale major surface protein (MSP)2 and Th1 adjuvants induced strong CD4 T cell proliferative and IFN-γ responses in cattle, that upon challenge were rapidly and permanently lost. To determine if A. marginale infection induced suppression or peripheral deletion of antigen-specific T cells, MHC class II tetramers were used to enumerate epitope-specific T cells following immunization of calves with a DNA construct encoding the 30-mer F2-5 epitope of MSP1a and infection six months later. As observed for MSP2, the MSP1a F2-5 peptide-specific CD4 T cell responses, still strong at the time of challenge, rapidly disappeared by 2–4 weeks. BoLA MHC class II DRB3 *1101 tetramers were constructed with the 15-mer core F2-5 epitope, designated F2-5B, linked to the DRB3 chain. Tetramer-stained PBMC obtained following immunization and challenge were selected with anti-PE beads prior to FACS analysis to increase the sensitivity of detection. The loss of MSP1a peptide F2-5-specific T cell responses was paralleled by a rapid decline in circulating DRB3 *1101-F2-5B tetramer-positive CD4 T cells after challenge, showing that the lack of response is not simply due to anergy or suppression. Our data support the hypothesis that the antigen-specific cells undergo activation-induced cell death in response to the infection, as tetramer-positive cells were not detected in spleen or liver.
CD4+CD25+ regulatory T cells attenuate hypersensitivity pneumonitis by suppressing IFN-γ secreting T cells (B25)
Authors: Park Yuna , Chung Doohyun
Published: January 2023

Hypersensitivity pneumonitis(HP) is developed after repeated inhalation of finely dispersed antigens. The pathogenesis of HP is complex and not completely understood. Currently, HP is mainly caused by Th1 immune response. CD4+CD25+ T cells play critical roles in preventing the development of various immune diseases. Therefore, we postulated that CD4+CD25+ T cells might contribute to attenuating HP.

To address this postulation, we investigated functional roles of CD4+CD25+ T cells in Saccharopolyspora rectivirgula (SR)-induced HP. CD4+CD25+ T cells infiltrated the lung tissue in B6 mice during the development of SR-induced HP. In CD4+CD25+ T cell-depleted B6 mice using PC61 mAb, HP was worse in terms of total cell number, SR-specific IgG in serum and bronchoalveolar lavage fluid (BALF) and SR-specific T cell proliferation than those in control B6 mice. The adoptive transfer of CD4+CD25+ T cells attenuated SR-induced HP in mice depleted CD4+CD25+ T cell. CD4+CD25+ T cell depleted mice showed elevated IFN-γ production and reduced IL-4 production in the lung during HP. Moreover, CD4+CD25+ T cell suppressed IFN-γ-producing CD4+ and CD8+ T cells in BALF with direct contact manner rather than indirect mechanism.

Taken together, these results suggest that CD4+CD25+ regulatory T cell play a protective role in SR-induced HP by suppressing IFN-γ-producing CD4+ and CD8+ T cell, which induced Th1 phenotype during the pathogenesis of HP. The enhancement of CD4+CD25+ regulatory T cell function may be useful therapeutic modality for HP.
Impaired T cell function and CD8 memory maintenance in a novel IL-7Rα knock-in mouse (B30)
Authors: Abraham Ninan , Dhanji Salim , Priatel John J , Miners M Jill , Teh Hung-Sia , Osborne Lisa C
Published: January 2023

Interleukin (IL)-7 is an essential growth factor for thymocyte development and survival of peripheral T cells, specifically CD8+ memory T cells. Current models such as IL-7 or IL-7 receptor alpha (IL-7Rα) knock-out mice have demonstrated the necessity of IL-7 signaling in these processes, but are unable to delineate the involvement of downstream pathways. To more precisely identify signals governing IL-7 function in vivo, we have disrupted the IL-7Rα Y449XXM motif in mice by knock-in mutagenesis (IL-7Rα449F). Thymocytes from these mice overcome a double negative (DN) stage developmental block that allows subsequent T cell maturation and peripheral migration. This is in stark contrast to the severe cytopenia seen in IL-7Rα−/− mice. These findings highlight the importance of IL-7Rα Y449-mediated signals during thymopoiesis, but also indicate a requirement for Y449-independent signals. Infection of IL-7Rα449F mice with the intracellular pathogen Listeria monocytogenes revealed that CD4 and CD8 T cells had different requirements for IL-7Rα signals. Strikingly, CD4 T cells failed to mount a detectable primary response to Listeria. Despite normal CD8 primary responses, maintenance of Listeria-specific CD8 memory was impaired in IL-7Rα449F mice. Furthermore, we show that Bcl-2 is IL-7Rα Y449-independent and insufficient for IL-7-mediated maintenance of CD8 memory.

Research funded by the Canadian Institutes of Health Research and the Michael Smith Foundation for Health Research
The effect of BLyS neutralization on primary B cell compartments and responses (B31)
Authors: Scholz Jean L. , Crowley Jenni E. , O’Neill Patrick J. , Cho Yun Hee , Ward Chris , Migone Thi-Sau , Cancro Michael P.
Published: January 2023

B lymphocyte stimulator (BLyS) controls the proportion of transitional B cells completing differentiation and the longevity of most primary B cells. These key roles in B cell selection, survival and homeostasis make BLyS and its receptors attractive candidates for targeted B cell therapeutics. Here we have used a neutralizing hamster anti-mouse BLyS antibody to assess how BLyS depletion influences developing and primary B cell subsets, as well as how this treatment impacts primary TD and TI immune responses. Mice treated with 10F4 show rapid and substantial reductions in the transitional, follicular, and marginal zone pools which persist for ~40 days. In contrast, the only bone marrow subset affected is the mature recirculating B cell fraction. Interestingly, splenic B1 cells, but not peritoneal B1 cells, are reduced as well. Following the recovery of serum BLyS levels, peripheral reconstitution occurs gradually, such that normal B cell numbers return by day 70–80. Mice challenged with T-dependent or T-independent antigen at day 30 post-treatment with anti-BLyS mount attenuated humoral responses compared with untreated mice. Together, our results show that BLyS neutralization ablates most primary B cells but spares B lineage progenitors; and indicate that these populations, as well as BLyS per se, may be required for unabridged primary immune responses.
Effect of human recombinant interferon alpha 2b (Viferon) in treatment of severe bacterial infections in neonates. (B32)
Authors: Soldatova Irina Gennadyevna , Volodin Nicolay N. , Malinovskaya Valentina V. , Degtyareva Marina V. , Vyzhlova Eugenia N.
Published: January 2023

Neonates have decreased cytotoxic mechanisms and low levels of endogenous interferon production. Aim: Clinical efficacy and mechanisms of adjunctive immunotherapy with human recombinant interferon alpha 2b (Viferon) were studied in neonates suffering from severe bacterial infections. Design. 48 neonates with bacterial pneumonia were observed in NICU. 23 infants (group 1) were treated with Viferon-1, 150,000 IU per suppositorium q8h per rectum during 7 days in addition to combined antibacterial and supportive therapy. 25 babies (group 2) received the same basic therapy plus placebo instead of Viferon in similar way. Results. Clinical course of pneumonia was more favourable in neonates treated with Viferon. The duration of central venous line usage and total parenteral nutrition were significantly less in group 1 than in group 2. Inflammatory signs in blood analyses were significantly decreased after the treatment with Viferon in comparison with placebo-group. Infants treated with Viferon had significantly higher levels of CD3+, CD4+ and CD25+-lymphocytes, higher serum levels of IFN, elevation of mitogen-induced IFN-gamma production after Viferon than infants from group 2. Conclusion. Immunotherapy with Viferon improves the clinical course of severe bacterial infections and restores the balance in immune system of infected newborn infants.
Development of PKC Theta-Specific Inhibitors to Modulate T-Cell Activation (B35)
Authors: Karim Felix , Baruch Amos , Cruz Jay Dela , Maclean Derek , Das Kanad , Harrision Stehen
Published: January 2023

Autoimmune diseases such as psoriasis and rheumatoid arthritis involve inappropriate activation and infiltration of T lymphocytes. Protein Kinase C Theta (PKC Theta) activation is a critical step in the integrated TCR/CD28 co-stimulatory signaling cascade leading to productive T-cell activation. Mice deficient in PKC Theta have been shown to have attenuated T-cell response and are protected from antigen-induced arthritis. Efforts to develop small molecule (ATP-competitive) inhibitors of individual PKC isozymes for therapeutic applications have been largely unsuccessful due to the lack of specificity of such compounds. KAI Pharmaceuticals has developed highly selective peptide-based inhibitors for each PKC isozyme that overcome this limitation by modulating the unique intracellular translocation of individual isozymes. KAI is actively developing PKC Theta inhibitors to treat and modify autoimmune diseases. The development and characterization of these highly selective PKC Theta inhibitors/modulators will be discussed.
Antigen-independent secondary Igκ rearrangement in B-cell development – implications for receptor editing (B39)
Authors: Liu Feifei , Cowell Lindsay G. , Heikamp Emily , Kelsoe Garnett
Published: January 2023

Receptor editing by secondary Vκ-to-Jκ rearrangements is thought to be driven by reactivity to self. However, the evidence supporting antigen-driven editing is open to other interpretations; analysis of Igκ excision circles from birds and mice indicates continuing V(D)J recombination in cis after both functional (F) and non-functional (nF) rearrangements. We have determined the extent and nature of secondary Vκ-to-Jκ rearrangements by analyzing their intermediate cleavage products in the 103/Bcl2 cell line and in purified compartments of developing B cells from normal, Jκ−/+, and IgH transgenic mice. In all cases, we find that the ratio of F:nF VκJκ joints replaced by secondary rearrangement is approximately 1:2, the ratio expected for rearrangement without feedback of any kind. Secondary rearrangement is initiated in small pre-B cells and in BrdU pulse-labeling studies, we detected no evidence for immature to pre-B “retrograde” differentiation. Thus, “receptor editing” occurs in B cells that do not express surface Ig. Significantly, in 3H9 IgH transgenic mice, replacement rearrangements exhibited F:nF ratios of 1:2, regardless of whether the initial rearrangements were permissive or non-permissive for autoreactivity. Our results demonstrate continuing Vκ-to-Jκ rearrangement on a single chromosome without feedback and imply that “receptor editing” is an illusion of antigen-driven selection.
The spatial-temporal interactions of CD45 with the B cell receptor and regulatory roles in intracellular signaling (B45)
Authors: Tam Anthony Waiyee , Tsourkas Philippos , Raychaudhuri Subhadip
Published: January 2023

Background: CD45, a membrane-bound phosphatase, has been shown to enhance and inhibit intracellular signaling in many different immune cells including B lymphocytes. However, the mechanisms of CD45 have not been fully described.

Objectives: To build a computational model for B cell signaling in order to investigate the role of CD45 in the initiation of intracellular signaling upon B cell receptor ligation with antigen and the spatial-temporal interactions with the B cell receptor and signaling protein tyrosine kinases.

Methods: Kinetic Monte Carlo stochastic simulations will be used with probabilistic rate constants mapped to experimentally measured parameters. Simulation results will be tested in vitro.

Anticipated results: It will be shown that without a binding partner, CD45 will be pushed out early in the formation of the B cell immunological synapse. For the duration that CD45 interacts with intracellular Src family kinases (SFK) in close proximity of clustered B cell receptors, there is sufficient activation of SFK to initiate the signaling cascade. It will also be shown that it is critical for CD45 to be pushed out from the immunological synapse away from BCR and the SFK so that non-phoshorylated BCR should be internalized and used for antigen capture and presentation on MHC II.

530 754-5739
Expression of type III lambda interferon genes is showing similar two-wave regulation with type I alpha/beta interferon genes (B50)
Authors: Osterlund Pamela , Pietila Taija , Kotenko Sergei , Julkunen Ilkka
Published: January 2023

In virus infection the replication of viral genome triggers the expression of antiviral genes, like type I interferons (IFN-alpha/beta), by transcription factors activated through both TLR-dependent and independent pathways. Recently, another class of antiviral type III interferons (IFN-lambda1-3) was identified. Here is presented a detailed characterization of the IFN-lambda1 and IFN-lambda3 gene promoters and functional characterization of virus-specific activation of transcription factors regulating these promoters. Both of these promoters have a functional interferon-stimulated response element (ISRE) and NF-kappaB binding site, although the interferon regulatory factors (IRFs)-binding ISRE was the most crucial regulatory element on these promoters. Ectopic expression of the components of the three virus-induced signaling pathways (MyD88+IRF7, Trif and RIG-I) induced the activation of IFN-lambda1 promoter, whereas the IFN-lambda3 promoter was activated to higher extent only by IRF7 alone or in combination with MyD88. Recently documented IRF3 suppressor Pin1 down-regulated all the three signaling cascades induced IFN promoter activation, but not the NF-kappaB mediated transcription. These results suggest that IFN-lambda1 gene is regulated by both IRF3 and IRF7 like IFN-beta gene, whereas IFN-lambda2/3 genes are induced mainly after IRF7-mediated positive feedback.
CPG potentiates the effects of cigarette smoke on releases of IL-8 in neutrophils (B55)
Authors: Mortaz Esmaeil , Adcock Ian , Ito Kazuhiro , Nijkamp Frans P
Published: January 2023

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide. Smoking is considered the major cause of the disease. All smokers develop airway inflammation through oxidative stress with macrophages, neutrophiles and mediators (such as IL-8) involved. Previous studies have implicated a role of bacterial DNA(CpG, in the initiation of inflammation. In this study we investigated the capacity of cigarette smoke extracts (CSE) and CpG-DNA to trigger and modulate IL-8 responses in human neutrophils. A human isolated neutrophils, were exposed to the CSE, CpG, or in combination. Supernatants were isolated and analyzed for IL-8 by ELISA. The NF-B and AP-1/c-Fos activities were monitored by Western blot by using antibodies. CSE and CpG induced the release of IL-8. When co-administered release of IL-8 synergically increased which was correspondence with degradation of IB- in cytoplasm. CpG and CSE evoked concomitant increases in intracellular NO levels and consequently, nuclear accumulation of c-Fos and NF-B. Pharmacological inhibition of NF-B activity or using of N-acetylcysteine (NAC) attenuated IL-8 release. These results identify reactive oxygen dependent activation of NF-B and c-Fos as an important mechanism to CSE and CpG. In conclusion, this study show that the combination of CSE and CpG provide a substantial release of IL-8 which may accounts to exacerbation of lung in COPD patients when they are exposed to bacterium infections.

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Intracellular signaling networks in the immune response: Interleukin-4 introduces competition and cooperation to interleukin-2-induced T cell proliferation (B57)
Authors: Comfort Kristen K , Haugh Jason
Published: January 2023

Cells sense and respond to stimuli through signal transduction pathways, which mediate proliferation, differentiation, and survival. The cytokines interleukin-2 (IL-2) and interleukin-4 (IL-4) are key regulators of the immune system, influencing the expansion and differentiation of T cells. Both synergistic and antagonistic effects of IL-2 and -4 co-stimulation have been shown; the antagonism may arise from the sharing of a common receptor subunit. We have sought to characterize IL-2 and IL-4 signaling at the level of intracellular pathways activated by these receptors. IL-2 receptors are known to activate the Ras/Erk and phosphoinositide (PI) 3-kinase pathways as well as the STAT5 transcription factor. IL-4 is able to activate PI 3-kinase/Akt as well as STAT6, though not Ras/Erk.

We found that IL-4 initially antagonizes, and later synergizes with, IL-2-stimulated HT-2 cell proliferation; a murine T cell line. At a signaling level, IL-4 abates IL-2-stimulated activation levels of Akt, Erk and STAT5 possibly through competition for limiting amounts of common receptor subunit. IL-2/IL-4 co-stimulation provokes prolonged activation only of STAT6. This extended STAT6 activation may be critical in the IL-2/IL-4 induced synergy in T cell growth. Currently, we are investigating the crosstalk between these pathways and their functional roles in IL-2 and IL-4-stimulated T cell responses.
Association of gender and IL-6R function and its role in alcoholic liver disease (B58)
Authors: Gallucci Randle M , Lee Eric G , Mickle Bethany M
Published: January 2023

Alcohol liver disease (ALD) is more severe in females than males. Proinflammatory cytokine release from Kupffer cells during inflammation is associated with the pathogenesis of ALD, and female chronically ethanol fed rats express cytokine mediators differently than male counterparts. Differences in IL-6 receptor function in females may play a role in the clinical observations of more severe ALD in females. In this study, the effects of sex steroid exposure on the expression and function of IL-6 receptor in ethanol fed rats were examined. Normal male and female or ovarectomized Wistar rats were fed liquid diet with and without ethanol for up to four weeks. Ovarectomized rats were additionally implanted with osmotic pumps containing either testosterone or estrogen. Ethanol consumption significantly increased in IL-6 receptor mRNA and protein expression and NFkB activation in female and estrogen treated rats. However C/EBPβ and IkB expression was higher in male and testosterone treated rats, indicating increased IL-6 signaling, and decreased NFkB signaling. Our data suggest that sex steroids play a role in IL-6R expression and function and that testosterone may be protective in ALD.
The Polycomb Group Member Bmi-1 Determines The Proliferative Potential Of CD8 T Cells (B6)
Authors: Heffner Maike , Fearon Douglas T.
Published: January 2023

In persistent viral infections certain virus-specific CD8+T cell clones demonstrate long term expansion despite evidence of cellular replicative senescence, indicating that within these clones are cells with stem cell properties to maintain continuous generation of effector cells. A candidate for maintaining the replicative function of these putative “stem cells”, is Bmi-1 which enables self renewal of haematopoietic and neural stem cells.

Stimulation of OT-I CD8+ T cells with 2.5nM SIINFEKL peptide upregulated Bmi-1 mRNA by 10 fold in 6 hours and protein by 24 hours. Expression was reversible upon removal of peptide, but was maintained by IL-2 and IL-15. Knockdown of Bmi-1 with a lentiviral vector expressing a specific shRNA impaired the proliferative capacity of IL-2 stimulated OT-I CD8+ T cells by 65% by day 4, and of CTLL-2 cells by 80% at day 12 after transduction. Ectopic expression of Bmi-1 in OT-I CD8+ T cells with a retroviral vector led to a 4.6 fold increase in cell number 10 days after transduction relative to cells with vector alone. OT-I CD8+ T cells adoptively transferred to Rag2−/−mice and expressing ectopic Bmi-1 expanded five-fold more than control OT-I CD8+ T cells after 80 days.

Thus, certain CD8+ T cells may resemble hematopoietic and neural stem cells in their expression of Bmi-1 to regulate replicative potential.
Resident Dendritic Cell (DC) Activation Initiates Early Innate Response to Kidney Ischemia-Reperfusion Injury (IRI) and Inflammation via IL-23/IL-17 and IL-12/IFN-γaxis (B72)
Authors: Li Li , Huang Liping , Ye Hong , Vergis Amy L , Sung Sun-Sang J , Lobo Peter I , Okusa Mark D
Published: January 2023

DCs contribute to innate immunity, autoimmunity and host defense through IL-12/IFN-γ and/or IL-23/IL-17 pathways. Although DC is the dominant kidney leukocyte subset, its role in the early IRI is unknown. We hypothesize that early kidney IRI is characterized by resident DC activation leading to IL-12/IFN-γ and IL-23 production. Depletion of DCs using lipsoomal clodronate prior to IRI led to kidney protection, which indicated the importance of DCs. DC triggered NKT cell IFN-γ production and its effect on kidney IRI was investigated by using αGalCer-primed BMDC. Adoptive transfer of BMDC-αGalCer or control BMDC into B6 mice was performed prior to kidney IRI. Plasma creatinine was 5-fold higher in DC-αGalCer treated IRI mice than those with BMDC (P<0.001). Furthermore, DC-αGalCer treated CD1d−/− or IFN-γ−/− mice have the minimal injury and similar to the BMDC treated mice. Next we observed that as early as 4–6 hrs post kidney IRI there was a >25-fold increase of IL-23p19 and IL-12p40 mRNA. Anti-IL-23p19 mAb protected kidneys from IRI and led to a reduction of PMNs and macrophages compared to control IgG. Lastly compared to WT mice, IL-12p35−/−, IL-12p40−/− mice kidney showed evidence of significant functional and histological protection (P<0.001). These results demonstrate that DC activation and down stream mediators play a pivotal role in the early kidney IRI and could serve as targets for new therapies.
Macrophage migration inhibitory factor is involved in the pathogenesis of antigen-induced arthritis in mice (B78)
Authors: Rodriguez-Sosa Miriam , Juarez-Avelar Imelda , Rivera Irma , Verdin Leticia , Terrazas Luis Ignacio
Published: January 2023

Rheumatoid arthritis (RA) is a chronic autoimmune disease caused mainly by pathogenic activation of T-cells recognizing cartilage antigens. Infiltration of these autoreactive T-cells and macrophages into the synovium results in the formation of pannus, eventually destructing bones and ligaments. MIF is a 12 kDa highly conserved trimeric protein which acts as an enzyme, hormone and cytokine. MIF is largely known for its role in macrophage effectors function and T cell activation, as well as a pro-inflammatory molecule. Using a murine antigen-induced arthritis model (AIA) in MIF−/− and MIF+/+ BALB/c mice we analyzed the role of endogenous MIF in rheumatoid arthritis. AIA was induced by intra-articular injection of heat aggregated OVA in presensitized (i.v. transferred CD4+ DO11.10 cells) mice. MIF −/− mice displayed low signs of disease, given that inflammation and eritema were reduced, these observations were associated with low levels of TNF-α, IFN-γ and nitric oxide in serum. Histological arthritic scores for synovial inflammation were also significantly decreased compared to MIF+/+ mice. Similarly, levels of MMP-2 and MMP-9 performed by inmmunohistochemistry, were lower. These results indicated that MIF plays a critical role in inflammation and joint destruction in AR.

Supported by DGAPA-PAPIIT IN208606-3.
Regulatory T cells selectively protect against experimental autoimmune encephalomyelitis induced with self but not foreign antigens (B79)
Authors: Akirav Eitan Moshe , Xu Yan , Bergman Cheryl M , Hill Myriam , Ruddle Nancy H
Published: January 2023

Naturally occurring CD4+CD25+ regulatory T cells (Treg) are selected in the thymus by self-antigens and can prevent the development of autoimmune diseases. Experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), can be induced with myelin oligodendrocyte glycoprotein (MOG). Here we show that both MOG mRNA and protein are detected in the thymus. To test whether Tregs specific for mouse MOG protect against EAE, we examined the effects of Treg depletion on EAE induced with the Ig-like domain of mouse (self), rat or human (foreign) MOGs. Treg depletion resulted in increased EAE severity following immunization with mouse MOG, but surprisingly, did not affect EAE induced with either rat or human MOG. The increase in disease severity following Treg depletion of mouse MOG immunized mice was associated with increased T cell proliferation and IL17 production as well as an increase in T cell response to the immunodominant peptide MOG35-55. In the central nervous system, T cells from mouse MOG immunized mice showed increased infiltration as well as increased ratio of IFNγ to IL10 producing cells following depletion. These data demonstrate the high specificity of Tregs to self but not foreign MOG, thus, underlining their inability to “cross-protect” against autoimmunity induced with closely related antigens.

Supported by NMSS RG2394 and NIH CA16885.
Massive Changes in the TCR Repertoire of Regulatory T Cell-Deficient Mice: Multiple Vb spectratype alterations indicate strong responses by a large repertoire of autoimmune T cells (B88)
Authors: zheng lingjie , sharma Rahul , deshmukh umesh s , Jarjour Wael N , Kung John , Fu Shu Man , Ju Shyr-te
Published: January 2023

B6.Cg-Foxp3sf/J mice, a B6 strain lacking functional Foxp3 transcription factor and CD4+CD25+Foxp3+ regulatory T (Treg) cells, spontaneously develop multi-organ autoimmune responses and die at 4 weeks old. We hypothesize that the autoimmune reactions in scurfy mice will change their TCR repertoire as compared with normal B6 mice. We observed no significant changes in their Vb family expression levels by staining with fluorescent anti-Vb mAbs. However, their Vb family spectratypes were remarkably different from that of B6 mice and from each other, whereas Vb spectratypes of individual B6 mice were highly similar to each other. Significant differences were found for all 20 Vb families examined. Significant spectratype changes in many Vb families were also observed in Treg cell-deficient IL-2 knockout (KO) and IL-2Ra KO mice. These data demonstrate that Treg cell-deficiency allows a strong polyclonal expansion of a large autoimmune T cell repertoire, resulting in massive changes in the TCR repertoire in many or all Vb families.
MERCURY-INDUCED ACCELERATION OF SPONTANEOUS AUTOIMMUNITY IN PRE-NEPHRITIC (NZBxSWR)F1 (SNF1) FEMALE MICE (B90)
Authors: Pilones Karsten A , Gavalchin Jerrie
Published: January 2023

Mercury exposure in susceptible mice has been known to induce autoimmunity, including the proliferation of autoantibodies, Th2-dominated antibody responses and immune complex disease. We investigated whether the immunostimulating effects of mercury can accelerate the induction of autoimmunity in SNF1 mice, a model that closely resembles human SLE nephritis. Previous studies from our laboratory have shown that dysregulation of the idiotypic network is key in the development of renal immune complex deposits in SNF1 females. In this study, six week-old pre-nephritic SNF1 females were exposed to 10 mg/L of either mercuric chloride or methylmercury in drinking water and examined for modulations in pathogenic immunophenotypes, autoantibody titers and clinical evidence of disease. We found increased populations of splenic T-cells and B-cells reactive with a pathogenic idiotypic determinant (IdLNF1) in mice exposed to mercury which, interestingly, also showed reductions in the percentage and absolute numbers of regulatory phenotypes (CD4+CD25+GITR+). Splenic lymphocytes from exposed mice showed increased proliferative responses to IdLNF1. LPS-induced proliferation was also increased in exposed mice, regardless of mercurial species used. We found modulations in serum antibody levels, including increased titers of anti-double stranded DNA autoantibodies. Clinically, mice exposed to mercury showed increased proteinuria and are currently being evaluated for glomerular damage and nephritis. The results of this study support the hypothesis that low-level exposure to metal xenobiotics such as mercury may trigger or accelerate autoimmune disease especially in a background of genetic susceptibility.
The role of PD-1/PD-L and B7/CD28 pathways in modulating anti-islet immune responses (B94)
Authors: Yadav Deepak , Hill Natasha , Yagita Hideo , Azuma Miyuki , Sarvetnick Nora
Published: January 2023

Costimulation via the PD-1/PD-L and B7/CD28 pathways modulates immune response towards self/non-self. We investigated whether the PD-1/PD-L pathway is impaired in autoimmune diabetes. Secondly, we hypothesized that B7-2 affects antigen presenting cell (APC) function. A progressive increase in the proportion of PD-1, B7-H1 and B7-DC positive T cells and APCs was observed in female non-obese diabetic (NOD) mice with age. However, NOD mice not only exhibited reduced availability of PD-1, B7-H1 and B7-DC signals as compared to non-diabetic C57BL/6 mice, but the capacity to induce B7-H1 expression was also reduced in NOD islets. In vivo blocking studies revealed that B7-H1 and B7-DC co-operate with B7-2 in augmenting priming and expansion of naïve anti-islet CD4 T cells. Lack of B7-2 significantly reduced the phagocytic ability and survival of dendritic cells. Interestingly B7-2 deficiency caused impaired PKC-epsilon response in APCs upon CD28-Ig stimulation. Thus, our data suggest that susceptibility to diabetes in NOD mice is associated with a defect in the availability of PD-1/PD-L mediated costimulation. On the other hand, B7-2 promotes APC function and survival.

D.Y. is recipient of JDRFI postdoctoral fellowship.

This work is supported by grants from NIH (# DK054063 and # AI51973) to N.S.
Mature B cells from healthy people which are permanently class switched to the IgD isotype are autoreactive (B95)
Authors: Koelsch Kristi A , Zheng Nai-Ying , Zhang Qingzhao , Duty Andrew , Abraham Leni A. , Helms Christina , Mathias Melissa D. , Jared Mathew , Smith Kenneth , Wilson Patrick C.
Published: January 2023

Determining the origin and fate of autoreactive B cells is critical to understanding and treating autoimmune diseases. We report that compared to antibodies from naïve and IgG memory B cells those from cells that have class switched to IgD via genetic recombination (Cδ-CS) are highly reactive with self-antigens, despite being derived from healthy people. Half of the antibodies from Cδ-CS B cells bind various autoantigens, and they are frequently polyreactive. Many also bind HEp-2 antigens. Intriguingly, some Cδ-CS B cells have accumulated residues in the variable regions that mediate anti-DNA reactivity via somatic hypermutation and selection, suggesting an immunological origin for anti-DNA antibodies in healthy people, while other Cδ-CS B cells are naturally autoreactive. We interpret these findings to indicate that autoreactive B cells can either be induced to class switch to IgD or autoreactive B cell clones that use IgD as the B cell receptor are not effectively deleted. Determining the mechanism by which the majority of Cδ-CS B cells are autoreactive may be important in understanding peripheral tolerance mechanisms and may provide insight to the enigmatic function of the secreted IgD antibody.
Parenteral Denaturization of Foreign Proteins
Authors: Boone T. H. , Manwaring W. H.
Published: January 2023

The accelerated denaturization of horse proteins in homologously hypersensitive dogs is due to some specific factor, the non-specific effect of sensitization being an inhibition or exhaustion of the normal, alien-protein denaturization function.
Physical forces can trigger T cell receptor signaling (35.10)
Authors: Roffler Steve R , Li Ya-Chen , Chen Bing-Mae , Wu Pei-Chun , Cheng Tian-Lu , Liao Yuan-Chun , Kao Lung-Sen , Tao Mi-Hua , Lieber Andre
Published: January 2023

Engagement of the T cell receptor (TCR) by antigenic peptides presented by the major histocompatibility complex (pMHC) activates specific T cells to control infections. The mechanism of TCR triggering, however, has remained obscure. Here, using micromanipulation of individual T cells, we show that physical forces acting on the TCR can induce calcium mobilization in a Lck-dependent manner. Investigation of artificial antigen-presenting cells (APCs) expressing engineered TCR ligands supports a model in which intercellular tension on the TCR generated during ligand engagement can initiate signaling in T cells. Furthermore, specific interactions between intercellular adhesion molecule-1 (ICAM-1) on APCs and the integrin LFA-1 on T cells may regulate tension to set the threshold for TCR triggering during ligand engagement.
Role of CD4 and CD8 coreceptors in the membrane proximal signaling and signal enhancement by self-peptide (35.15)
Authors: Artomov Maksym , Chakraborty Arup K
Published: January 2023

Current biophysical measurements of the coreceptor/MHC binding kinetics present a puzzle when trying to reconcile conventional understanding of coreceptor as a recruiter of the Lck to engaged TCR with the unusually low stability of MHC-coreceptor interaction (koff >20 s-1). Moreover, the fast kinetics of MHC-coreceptor interactions is in direct contradictions with the assumptions of the currently available models of signal enhancement by non-cognate peptides when presented in mixture with small amounts of stimulating peptides. In our study we describe how competition between the diffusion of proteins in the membrane and their mutual interactions creates the spatial organization of membrane proteins (T-cell receptor, coreceptor, MHC protein) leading to successful signaling. Using spatially resolved computer simulations, we find, indeed, that fast kinetics of coreceptor-MHC interaction is of critical importance for the maintenance of the proper balance between diffusion and protein interactions.

The mechanisms unveiled in this study provide possible resolution to the important debate regarding the effect of coreceptor on the enhancement by self-peptides originated through the contradicting results obtained in CD4 and CD8 T-cells. We provide the signaling model explaining the difference in CD4 and CD8 T-cell in a single framework.
LFA-1 regulates CD8+ T cell activation through both TCR-mediated and LFA-1-mediated Erk1/2 signal pathways (35.16)
Authors: Ma Qing , Li Dan , Molldrem Jeffrey J
Published: January 2023

LFA-1 regulates T cell activation and signal transduction through the immunological synapse. TCR stimulation rapidly activates LFA-1, which provides unique LFA-1-dependent signals to promote T cell activation. However, the detailed molecular pathway and the precise mechanism by which LFA-1 contributes to TCR activation remain unclear. Herein, we report that LFA-1 directly participates in Erk1/2 signaling upon TCR stimulation in CD8+ T cells. The presence of LFA-1, not ligand binding, is required for the TCR-mediated Erk1/2 signal pathway. LFA-1-deficient CD8+ T cells have defects in sustained Erk1/2 signaling and TCR/CD3 clustering, which subsequently prevents MTOC re-orientation, cell-cycle progression and mitosis. LFA-1 regulates the TCR-mediated Erk1/2 signal pathway in the context of immunological synapse for recruitment and amplification of Erk1/2 signal. Furthermore, LFA-1 ligation with ICAM-1 generates an additional Erk1/2 signal, which synergizes with the existing TCR-mediated Erk1/2 signal to enhance T cell activation. The LFA-1-mediated Erk1/2 signal pathway is independent of ZAP-70 and PI3K but partially depends on Lck. Thus, LFA-1 contributes to CD8+ T cell activation through two distinct signal pathways. The function of LFA-1 is to enhance TCR signaling through the immunological synapse and deliver distinct signal in CD8+ T cell activation.
Cytolytic synapses control effectiveness of target cell destruction by CTL (35.17)
Authors: Sykulev Yuri , Beal Allison M , Anikeeva Nadia , Varma Rajat , Cameron Thomas O , Norris Philip , Dustin Michael L
Published: January 2023

We have investigated the stability of cytolytic synapses as well as patterns of cytolytic granule polarization and kinetics of their release by CTL with different potency to kill target cells. Less potent CTL form unstable cytolytic synapses, accounting for a third of the difference in potency providing the best evidence that stable synapse is important for cytotoxicity. To determine what other unknown mechanistic components may account for the distinct potency of target cell killing, we evaluated differences related to various pathways of granule delivery to the secretory domain of these CTL. Visualization of lytic granule delivery shows that the granules can take long or short paths to the secretory domain. The difference in path is dictated by the kinetics of early TCR signaling. Rapid and robust early signaling causes swift granule concentration near the MTOC and subsequent delivery directly to the secretory domain - the shortest and fastest path. Indolent signaling leads to late recruitment of the granules that move first to the periphery of the synapse and then move tangentially to fuse at the outer edge of the secretory domain - a longer path. The short pathway of granule delivery is associated with more efficient killing. Thus, rapid granule delivery to the secretory domain and containment of released granules within a stable synapse determines the potency of CTL to destroy target cells.
pMHC ligand anchored on a plastic surface via a 61 nm flexible poly(ethylene glycol) linker triggers the TCR: implications for the mechanism of TCR signal initiation (35.18)
Authors: Ma Zhengyu , Janmey Paul , Finkel Terri H
Published: January 2023

A critical step in the immune response is signal initiation from the T cell antigen receptor (TCR) by its ligand, peptide antigen presented by the major histocompatibility complex (pMHC). How TCR is triggered by ligand binding is still unclear. An interesting aspect of the puzzle is the sharp contrast between the inability of soluble pMHC to trigger TCR and the high triggering efficiency of surface-anchored pMHC. Here, we investigate the boundary between soluble and surface-anchored pMHC by anchoring pMHCs on plastic surfaces via poly(ethylene glycol) (PEG) linkers of varying length. We found that pMHCs anchored on the surface via a 61 nm linker trigger TCR as efficiently as a much shorter 5.6 nm linker. The lack of negative impact on TCR triggering by the long linker does not support the kinetic segregation model of TCR triggering, in which the critical factor is the shortness of the pMHC-TCR bridge between the membranes of T cell and antigen presenting cell (APC). Our result supports the idea that TCR triggering does not require receptor cross-linking, since the length of the linker far exceeds the distance required for two pMHC molecules to cross-link the TCR. Furthermore, this result is consistent with the receptor deformation model - a long linker may be tolerated as long as the T cell and APC membranes detach to a distance greater than the length of the pMHC-TCR bridge, such that mechanical force can still be applied to the TCR.

Work supported by NIH R21 AI078387, Joseph L. Hollander Chair, and Joseph Stokes Jr. Res. Inst.
T cell raft clustering during T cell surveying of APCs requires CD28 (35.2)
Authors: Van Komen Jeffrey S , Mishra Sudha , Byrum Jennifer , Rodgers William
Published: January 2023

T cell activation requires the orchestrated restructuring of its plasma membrane rafts to form the immunological synapse (IS). Our published data show an antigen (Ag)-independent membrane raft cluster that concentrates the TCR and CD4 at the site of Ag presentation before TCR triggering. We term this event raft clustering during surveying (RaCS). However, the mechanisms by which RaCS occurs is unknown. Our recent experiments show RaCS is initiated by CD28-dependent mechanisms. For example, RaCS was not detected in CD28-deficent T cells. Experiments with CD28-deficient T cells transduced with genes encoding mutated CD28 molecules showed that both the PI3K- and Lck-binding sites of CD28 are necessary for RaCS. These results in conjunction with imaging experiments using PP2 or wortmannin treated T cells suggest that PI3K- and Lck-association with CD28 is required for RaCS. We also observed that RaCS is inhibited in anergic T cells, whereas, SLE model mice exhibited increased RaCS. Collectively, our results show that actin-dependent RaCS at the APC is one of the earliest events in T cell activation, and provides a mechanism for tuning downstream Ag-dependent signaling events. Future experiments will determine the mechanism by which CD28 activation signals cluster the T cell rafts, and the effect of RaCS on interactions within the TCR complex.

This work is supported by the American Cancer Society (PF-08-178-01-LIB to JVK) and the NIH (P50 RR015577 and R01 GM070001 to WR).
Inhibition of activation-induced T cell death by AIP2-mediated ubiquitination of EGR2 (35.20)
Authors: Fang Deyu , Chen An , Lee Sang-Myeong
Published: January 2023

E3 ubiquitin ligases, which target specific molecules for proteolytic destruction, have emerged as key regulators of immune functions. Several E3 ubiquitin ligases, including c-Cbl, Cbl-b, GRAIL, Itch, and Nedd4, have been shown to negatively regulate T-cell activation. Here we report that the HECT-type E3 ligase, AIP2, positively regulates T-cell activation. Ectopic expression of AIP2 in mouse primary T cells enhances their proliferation and IL-2 production by suppressing apoptosis of T cells. AIP2 interacts with and promotes ubiquitin-mediated degradation of EGR2, a zinc finger transcription factor that has been found to regulate Fas ligand (FasL) expression during activation-induced T cell death. Suppression of AIP2 expression by small RNA interference upregulates EGR2 and FasL expression and enhances the apoptosis of T cells. Therefore, AIP2 regulates activation-induced T cell death by suppressing EGR2-mediated FasL expression via the ubiquitin pathway.
Differential TCR signal transduction pathways involved in high and low avidity CD8+ T-cell responsiveness (35.26)
Authors: Sharma Sharad K , Alexander-Miller Martha A
Published: January 2023

CTLs that display high functional avidity are known to be optimal for pathogen clearance. However, the molecular mechanism that allows high avidity CTLs to respond at very low levels of peptide antigen remains to be elucidated. To investigate this question, we compared TCR signal transduction events in high and low avidity CD8+ T-cells generated from OT-I TCR transgenic mice. Upon stimulation with APCs preloaded with a range of Ova257-264 peptide concentrations (10^-6, 10^-9 and 10^-12 M), we found that high avidity CD8+ T-cells induced higher levels of MAPK ERK-1/2 phosphorylation as well as higher levels of the dephosphorylated form of NFAT at each peptide concentration used for stimulation. We next determined whether the observed divergence in NFAT activation was reflected in differences in calcium mobilization. We found efficient mobilization of Ca2+ in high avidity cells even when the lowest concentration of peptide was used for stimulation, a level of peptide at which no increase in calcium was observed in low avidity cells. The decreased peptide requirement for calcium mobilization in high avidity cells correlated with a decreased peptide requirement for ZAP-70 activation and CD3ζ phosphorylation, the latter of which was higher and prolonged in high versus low avidity cells. Thus the functional avidity of the CD8+ effector cells studies here is controlled by differential regulation of the most membrane proximal event, phosphorylation of CD3ζ. Studies are currently underway to determine the mechanism by which high avidity cells acheive CD3ζ phosphorylation at limiting peptide levels.
Calculations show that T cell antigen discrimination requires multiple localized binding events (35.3)
Authors: Dushek Omer , Aleksic Milos , van der Merwe Anton , Coombs Daniel
Published: January 2023

T cells of the adaptive immune system rapidly scan the surface of antigen-presenting-cells (APC) for foreign material, in the form of peptide-MHC, using their T cell receptors (TCR). T cell activation via TCR-pMHC binding is extremely sensitive (a single presented pMHC can elicit a transient response), specific (a single amino acid substitution on a presented peptide can dramatically alter the response), and the decision to respond (e.g. by forming an immune synapse) occurs rapidly. Mathematical modeling based on statistical distributions reveals that pMHC must be repeatedly sampled (by binding TCR) in order for the T cell to make the decision to respond (or not) reliably. We find that TCR clustering and coreceptors act to immobilize pMHC which ensures that multiple binding events arise from a given pMHC. Functional assays of 1G4 T cells binding to a panel of 17 altered-peptide-ligands (NY-ESO) are used to support predictions from the model. We propose that sensitive, specific, and rapid antigen discrimination that is reliable can take place between a single TCR/pMHC.
Comparison of Dexamethasone and Cyclosporine to selective protein Kinase C-theta inhibitor effects on blood IL-2 production and T cell activation (35.32)
Authors: Ho Melisa W.-Y. , Deng Bijia , Benoit Stephen , Chen Joan , Wang Daniel , Boschelli Diane , Goldman Samuel , Kasaian Marion , Chaudhary Divya
Published: January 2023

Protein kinase C-theta (PKCθ) is critical in T cell IL-2 production upon TCR stimulation, making PKCθ an attractive drug target for inflammatory diseases. Our goal was to compare the effects of PKCθ selective blockade to alternative immune modulators dexamethasone (Dex) and cyclosporine A (CsA). Compound A, a selective PKCθ inhibitor, was evaluated on T cell responses following co-stimulation in vitro. With pre-activated CD4+ T cells, both Compound A and CsA reduced IL-2 production with comparable potency. However, CsA inhibited T cell proliferation with 10 fold higher potency than Compound A. Dex was the least potent on T cells but was effective in blocking IL-2 production in human whole blood. Next we examined whether exposure to Compound A or CsA in a primary activation would affect the ability of T cells to respond to secondary TCR stimulation. Both Compound A and CsA reduced T cell proliferation upon re-stimulation. Exogenous IL-2 restored the proliferation for Compound A exposed T cells, but not for CsA exposed T cells. We confirmed that Compound A-treated T cells had higher CD25 expression relative to those treated with CsA, accounting for their greater IL-2 responsiveness. Taken together, these findings indicate that targeting PKCθ impairs T cell IL-2 production, but not IL-2 responsiveness, suggesting that this inhibitor may selectively modulate T cell responsiveness under specific activation conditions.