bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure-function analysis of the determinants of CD46 polarization in T cells-combined role for polarity proteins,lipid rafts and ERM proteins (87.19)
Authors: Pasam Anupama , Ludford-Menting Mandy J , Sacirbegovic Faruk , Russell Sarah M
Published: January 2023

CD46 is a ubiquitously expressed human cell surface protein that acts as a receptor for complements for various pathogens including the measles virus. We demonstrated that ligation of the immunoregulatory cell surface receptor, CD46, altered T cell polarity and impaired activation and effector function in response to TCR or NK cell receptor signalling. However the molecular mechanisms by which T cell function is inhibited are not known.

We have previously shown that CD46 binds to the polarity protein, Discs large (Dlg), and that this interaction is important for the polarized localization of CD46. Specifically, CD46 localizes to the uropod of T cells, and to the distal pole of T cells undergoing antigen presentation. Polarization of CD46 is partially reduced by mutation of the Dlg-binding site, and is also partially reduced by mutation of the Cysteine residue in the transmembrane domain that is palmitoylated to allow association of CD46 with lipid rafts. However, comparison of CD46 mutants suggests that other determinants are also important in CD46 polarization. We hypothesize that the ERM proteins interacts with CD46, regulates its polarization in T cells, and perhaps play a role in CD46 signal transduction. To assess this we have generated mutations in the ERM-binding consensus sequences of CD46 and expressed these mutants in a uropod-containing T cell line for functional characterization. These studies will help to elucidate the molecular mechanisms by which CD46 exerts its immunoregulatory effects.
Regulation of Bcl10 by serine 231 (87.2)
Authors: Kane Lawrence P. , Delmastro Meghan , Jordanhazy Ryan
Published: January 2023

Bcl10 is a component of the “CBM” complex of proteins, along with CARMA and MALT1. This complex regulates activation of the NF-κB-inducing IKK kinase complex, and is important for integrating signals from antigen receptors and co-receptors on T and B lymphocytes. We previously showed that activation of NF-κB by the Akt kinase in T cells requires an intact CBM complex, and that Akt may interact with - and regulate the phosphorylation of - one or more members of the complex. Using the online ScanSite algorithm, we identified serine 231 in Bcl10 as a possible site of Akt phosphorylation. We generated a form of Bcl10 in which serine 231 has been mutated to alanine (S231A), and assessed effects on Bcl10 function. Thus, the S231A form of Bcl10 activates the NF-κB pathway much less efficiently in T cells, compared with the wild-type protein. Paradoxically, NF-κB activation by S231A Bcl10 is enhanced in fibroblasts. In both cell types, expression levels of S231A Bcl10 are consistently lower than the wild-type protein. Finally, we have examined the role of S231 in the regulation of Bcl10 localization. However, in contrast to its effects on NF-κB activation, mutation of S231 appears to have no impact on sub-cellular localization of Bcl10. These results suggest that phosphorylation of Bcl10 at S231 is important for regulation of NF-κB and that this pathway may operate differently in T cells and fibroblasts.

Supported by the Ovarian Cancer Research Fund.
Rac GTPases are required for restimulation-induced CD4+ T Cell Death (RICD) (87.20)
Authors: RAMASWAMY MADHU , DUMONT CELINE , MUPPIDI JAGAN R , TYBULEWICZ VICTOR L , SIEGEL RICHARD M
Published: January 2023

In activated CD4 T cells, TCR restimulation or antigen leads to FasL mRNA synthesis, expression and eventually apoptosis through cis or trans FasL-Fas interactions, in the process known as restimulation-induced cell death (RICD). Apoptosis requires both FasL synthesis and secretion and a protein-synthesis independent ‘competency to die’ signal that sensitizes TCR-stimulated cells to apoptosis induced by FasL. Recent work in our lab correlated competency signals with translocation of Fas into specialized membrane micro-domains (lipid rafts) in TCR restimulated T cells. However, what signaling pathways underlie the TCR-induced ‘competancy to die’ signal is not known. We have now identified the small Rho GTPase Rac as a modulator of TCR signals that mediate competency. SiRNA knockdown of Rac1 in jurkats and human CD4 T cells, results in specific abrogation of TCR induced ‘competency to die’ via Fas. Transfection of constitutively active Rac1 rescues TCR mediated Fas apoptosis in Vav-1 deficient Jurkat cells, which are devoid of competency signals. Absence of Rac1 does not affect a number of other parameters of early T cell signaling, however, we observe reduction in dephoshorylation of the Ezrin-Radixin-Moesin (ERM) proteins. CD2Cre Rac1 flox/flox mouse T cells as well as Rac2 −/− T cells have deficient Restimulation Induced cell death (RICD), which is more severe in T cells lacking both Rac1 and Rac2 (Rac1−/+/ Rac2 −/−). Further, in Jurkat and mouse T cells, Rac1 deficiency inhibits FasL upregulation upon TCR stimulation. Therefore, Rho GTPase proteins are critically involved in two distinct aspects of restimulation-induced T cell apoptosis and may be a novel target for immunomodulatory therapy.
The Gads adaptor protein regulates proliferation and differentiation of TCRβ+ CD4−CD8− double negative thymocytes (87.3)
Authors: Yankee Thomas M. , Zeng Ling , Dalheimer Stacy L.
Published: January 2023

TCRβ is the first TCR chain expressed and can be detected for the first time at a stage of T cell development that precedes expression of CD4 and CD8. TCRβ expression triggers signaling pathways that promote survival, proliferation, and differentiation into CD4+CD8+ double positive (DP) thymocytes. Despite the importance of survival, proliferation, and differentiation of TCRβ+ CD4−CD8− double negative (DN) thymocytes, the signaling pathways regulating these outcomes are unclear. To investigate the signaling pathways that regulate cell fate in TCRβ+ DN thymocytes, we are using a mouse line that lacks expression of the Gads adaptor protein. Gads is required for optimal TCR-mediated calcium mobilization and Gads−/− mice have a significant defect in T cell development at the DN to DP transition (J. Immunol. 173: 1711). Here, we extended these results by examining the role of Gads in the survival, proliferation, and differentiation of TCRβ+ DN thymocytes. We found that Gads regulates survival and proliferation of TCRβ+ DN3L thymocytes, but not TCRβ+ DN3E cells. In addition, using an in vitro differentiation assay, we found that Gads−/− DN thymocytes differentiated more readily into DP thymocytes than wild-type cells. These data support a model in which Gads regulates the number of cell divisions triggered by TCRβ expression.

This research is supported by NIH grant P20 RR016443.
T-cell activation requires mitochondrial translocation towards the immunological synapse (87.32)
Authors: Schwindling Christian , Quintana Ariel , Wenning Anna Sylvia , Becherer Ute , Rettig Jens , Schwarz Eva C. , Hoth Markus
Published: January 2023

Activation of the adaptive immune response requires the interaction between antigen-presenting cells and T-cells. This cell-cell interaction, called the immunological synapse (IS), facilitates the activation of several T-cell receptor (TCR)-mediated signalling cascades including a rise in the cytosolic Ca2+ concentration ([Ca2+]i) through the activation of CRAC/ORAI1 channels. These channels are opened after depletion of intracellular Ca2+ stores and inactivated by the inflowing Ca2+ itself. We show by epifluorescence, 2-photon and total internal reflection microscopy, that a large fraction of mitochondria was moved to the immediate vicinity of the IS, a process highly dependent on the actin cytoskeleton. Mitochondrial movement to the IS was required to sustain the CRAC/ORAI1-mediated Ca2+ influx. Disruption of the actin cytoskeleton prevented mitochondrial movement and subsequent [Ca2+]i rises. The increased [Ca2+]i signals following interaction of the IS with mitochondria correlated with an enhanced T-cell proliferation. Our results show that actin cytoskeleton-mediated movement of mitochondria into the vicinity of the IS is required to sustain Ca2+ influx and permit efficient T-cell activation.

This project was funded by the Deutsche Forschungsgemeinschaft (SFB 530 and GRK 845) and a grant from the Saarland University (HOMFOR).
The X-linked lymphoproliferative disease gene product SAP associates with PIX and participates in T cell activation (87.37)
Authors: Wu Jun
Published: January 2023

SLAM (signaling lymphocyte activation molecule)-associated protein (SAP) is a SH2 domain-containing adaptor expressed in T cells and natural killer cells. Its essential role in immune responses is underscored by the recent finding that mutations in SAP result in a rare but fatal X-linked lymphoproliferative disease (XLP). Although SAP is known to associate with SLAM-family receptors, the exact molecular mechanism by which SAP regulates lymphocyte signaling remains elusive. We here report that in T cells, SAP associates with the PAK-interacting exchange factor (PIX), a guanine nucleotide exchange factor specific for Rac/Cdc42 GTPases. Moreover, SAP, PIX, and an activated form of Cdc42 form a complex in mammalian cells. We demonstrate that the SAP-PIX interaction is specific and is mediated by the C-terminal region of the SAP SH2 domain and the PIX SH3 domain. We further show that SAP is required for the recruitment of PIX to the SLAM-family receptors. Interestingly, over-expression of SAP, but not its homolog EAT-2, leads to a synergistic activation of NFAT in combination with a calcium signal in T cells. This SAP-mediated activation appears to be receptor-dependent and can be blocked by a dominant negative form of PIX. Taken together, our data strongly suggest that, in addition to the known SAP-interacting kinase Fyn, PIX may be another key player in SAP-mediated T cell activation.
The Requirement of Reversible Cysteine Sulfenic Acid Formation in T Cell Activation and Function (87.40)
Authors: Michalek Ryan D , Nelson Kimberly J , Yi John S , Stridiron Daya , King S Bruce , Poole Leslie B , Grayson Jason M
Published: January 2023

Reactive oxygen intermediates (ROI) generated in response to receptor stimulation play an important role in mediating cellular responses. We have examined the importance of reversible cysteine sulfenic acid formation in naïve CD8+ T cell activation and proliferation. We observed that within minutes of T cell activation, naïve CD8+ T cells increased ROI levels in a manner dependent upon antigen concentration. With increased ROI, levels of cysteine sulfenic acid were also elevated. To examine the contribution of reversible cysteine sulfenic acid formation to T cell activation increasing concentrations of 5, 5 dimethyl-1,3-cyclohexanedione (dimedone), which covalently binds to cysteine sulfenic acid, were added to cultures. Subsequent experiments demonstrated that the reversible formation of cysteine sulfenic acid was critical for ERK1/2 phosphorylation, calcium flux, cell growth, proliferation, and cytokine production of naïve CD8+ and CD4+ T cells. We also found that TNF-α production by effector and memory CD8+ T cells was more sensitive to inhibition of reversible cysteine sulfenic acid formation than IFN-γ. Together, these results demonstrate that reversible cysteine sulfenic acid formation is an important regulatory mechanism by which CD8+ T cells are able to modulate signaling, proliferation, and function.

Research supported by American Cancer Society- #RSG-04-066-01-MBC to Jason Grayson.
A molecular analysis of diminished expression of regulatory subunit β protein of type I protein kinase A in SLE T cells (87.45)
Authors: Giri Banabihari , Das Maitreyee , Grayson Jason M. , Khan Islam U.
Published: January 2023

The deficiency of type I protein kinase A (PKA-I) isozyme in systemic lupus erythematosus (SLE) T cells is a signaling disorder and occurs with a prevalence of 80%. Our earlier studies established that SLE T cells have a profound deficiency of regulatory subunit β of PKA-I (RIβ) protein. We hypothesized that the reduction of RIβ-subunit protein is due to altered post-transcriptional processing and/or instability of RIβ mRNA in SLE T cells. To test our hypothesis we studied the translational efficiency of RIβ mRNA in T cells in SLE patients and healthy subjects. CD3+ T cells from human blood were enriched by negative selection with magnetic microbeads. The polysomal fractions were isolated from cycloheximide-treated T cells on linear sucrose gradient. Total RNA was extracted from each polysomal fraction followed by RIβ mRNA quantification by quantitative real-time PCR (Q-PCR). The PCR analysis of polysomal fractions revealed that the polysome profile of SLE T cells was consistent with a global translational attenuation, and specifically a paucity of polysome-bound RIβ mRNA. Interestingly, the stimulation of SLE T cells with a combination of anti-CD3 and anti-CD28 antibodies restored the RIβ mRNA profile, consistent with active translation. In conclusion, reduced RIβ-protein expression in SLE T cells is the consequence of the instability of RIβ mRNA and its absence in high molecular weight polysomes.

Supported by NIH RO1-AR39501.
The role of non-stimulatory MHC-peptides in aiding antigen recognition by CD8+ T cells (87.49)
Authors: Gascoigne Nicholas R.J. , Yachi Pia P. , Lotz Carina , Ampudia Jeanette
Published: January 2023

T cells are able to identify minute quantities of antigenic MHC-peptide (MHCp) complexes among a sea of endogenous MHCp presented on an antigen presenting cell. We previously showed that non-stimulatory endogenous peptides can enhance T cell sensitivity to limiting quantities of antigen. Here we extend these findings to different T cell populations, and show that non-stimulatory peptides are most important in antigen recognition by less differentiated cells, such that their ability to enhance antigen recognition is the best in thymocytes, moderate in naïve T cells, and mild in effector T cells. The increased sensitivity to antigen in the presence of non-stimulatory peptides can be seen in the level of effector functions such as cytokine production and killing of target cells, as well as by phenotypic maturation markers, such as CD25, CD69, CD44, CD5 and HSA. The importance of non-stimulatory peptides is inversely proportional to the agonist activity of the stimulatory peptide presented. Each of the endogenous non-stimulatory peptides tested showed similar ability to aid in antigen recognition. This may indicate that the limiting factor in the ability of non-stimulatory MHCp to aid recognition of a class I-restricted antigen is the CD8-MHC interaction, rather than the strength of the TCR-MHCp interaction, which seems to be paramount in class II-restricted systems.

Supported by NIH GM065230.
IFN-γ secretion is polarized at effector immunological synapses (IS) in vivo. (87.52)
Authors: Lowenstein Pedro R , Barcia Carlos , Liu Chunyan , Wawrowsky Kolja , Kroeger Kurt M , Castro Maria G
Published: January 2023

In vitro cytokine secretion by T cells is polarized. For example, IFN-γ secretion is focused towards the immunological synapse (IS), while secretion of TNFα is multi-directional. Whether polarized cytokine secretion occurs in vivo is unknown. We studied the intracellular distribution of IFN-γ within T cells establishing IS with infected astrocytes in vivo. Astrocytes were infected with an adenovirus (Ads) expressing Thymidine Kinase (TK), a marker for infected cells. 30 days later we induced a systemic immune response against Ads, and 14 days after the immunization animals were sacrificed and brains studied by immunohistochemistry and confocal microscopy. Brain sections were stained for TCR, LFA-1, TK and IFN-γ. Confocal analysis revealed that IFN-γ is polarized in T cells establishing close anatomical contacts with Ad infected cells. Polarization was found in cells with or without polarized LFA-1 or TCR. This demonstrates that in vivo IFN-γ is selectively polarized towards the APC, and further suggests that maturation of the IS is not necessary for IFN-γ polarization. In consequence, polarized IFN-γ secretion may precede the maturation of immunological synapses.

This work was funded by grants from NINDS, NIH.
CD8+ T Cells Possess the Potential to Differentiate into Both a High or Low Avidity Effector Cell (87.7)
Authors: Kroger Charles J. , Alexander-Miller Martha A.
Published: January 2023

During an immune response, CTL clones are present that exhibit a wide spectrum of peptide sensitivities. While the property of functional avidity has been well documented, the range of mechanism(s) by which this T cell property can be regulated is unknown. For example, an unresolved question is whether distinct naïve T cell subsets exist that display “inherent” differences in their peptide requirements for activation, i.e. functional avidity. In this scenario distinct naïve T cell clones would selectively respond upon encountering a high vs. low antigen concentration. Alternatively, differences in peptide sensitivity could be an “induced” property within the responding CTL population following encounter with an APC. Although the latter model is consistent with data from previous studies, formal demonstration that a single clone can give rise to both high and low avidity daughter cells is lacking. Using a TCR transgenic model in which both high and low avidity CTL can be generated, we now demonstrate that naïve T cells do not exhibit differences in the amount of peptide required for conjugate formation and subsequent proliferation/activation. Furthermore, we show that individual T cell clones are capable of differentiating into either a high or low avidity CTL. These results provide the first formal demonstration that an individual cell can give rise to both high and low avidity progeny, suggesting that avidity modulation at the level of an individual cell may be an important contributor to the functional avidity of the CD8+ T cell response generated in vivo.

This work was supported by National Institutes of Health grant R01 AI43591 to M.A.A-M.
The dynamic equilibrium between dimeric and monomeric cell surface B7-1 regulates T cell activation and CD28 expression (88.1)
Authors: Bhatia Sumeena , Almo Steven C , Nathenson Stanley G , Hodes Richard J
Published: January 2023

Numerous studies have identified differences between B7-1:CD28/CTLA-4 and B7-2:CD28/CTLA-4 complexes that form at the immunological synapse. Our earlier studies have established that B7-1 is expressed as a mixture of predominant non-covalent dimers and monomers on the cell surface. In order to test the hypothesis that altering the nature of B7-1:CD28 complexes will have functional consequences for T cell activation, we generated a disulfide-linked covalent dimer of B7-1 (B7-1:Cys), as well as mutants of B7-1 (L58D and I68D) that exist as a monomer on the cell surface. We used modulation of cell surface CD28 to assess the outcome of binding of B7-1 mutants to CD28 as one measure of the dynamics of B7-1:CD28 interaction since earlier studies have shown that CD28 is transiently downregulated upon binding to WT B7-1. As expected, L58D and I68D mutants resulted in reduced T cell activation and reduced CD28 downregulation when compared to WT B7-1, potentially due to low avidity interaction between monomeric B7-1 and monovalent CD28. Surprisingly, B7-1:Cys also resulted in less T cell activation than WT B7-1. Moreover, B7-1:Cys also resulted in reduced downregulation of CD28. These data suggest a novel model in which optimal B7-1 function for CD28 downmodulation and T cell activation requires CD28 engagement by non-covalent B7-1 dimers, followed by dissociation of B7-1 to a monomeric state, potentially facilitating disengagement from CD28.
Reciprocal NFAT1 and NFAT2 nuclear localization in anergic T cells is determined by sub optimal calcium signaling (88.15)
Authors: Srinivasan Mathangi , Frauwirth Kenneth
Published: January 2023

Impaired calcium signaling is a hallmark of T cell anergy. Calcium signaling induces nuclear translocation and activation of the NFAT transcription factors by activating the NFAT phosphatase calcineurin. Using an in vivo anergy model, we found that anergic T cells showed defective nuclear localization of NFAT1 after stimulation compared to responsive cells. NFAT2 remained cytosolic in responsive T cells, but showed nuclear localization after stimulation of anergic cells. To understand the mechanism underlying the translocation pattern, we stimulated naïve T cells in the presence the calcium chelator EGTA. NFAT1 was detected in the nucleus at complete extracellular calcium concentrations, but when calcium levels were reduced with EGTA, NFAT2 was nuclear, while NFAT1 was cytosolic. This pattern of translocation was also seen upon cyclosporin treatment, at concentrations that cause partial inhibition of calcineurin. These data suggest that the differential translocation regulation of NFAT1 and NFAT2 in anergic T cells can be correlated to the defective calcium signaling during anergy. This implies that the gene transcription profile of NFAT1 and NFAT2 in anergic T cells could be regulated differently from responsive cells. Ongoing studies are directed at studying transcriptional activity of NFAT1 and NFAT2 in anergic T cells.
GITRL-mediated costimulation in CD25−conventional and CD25+ regulatory CD4 T cells (88.16)
Authors: Igarashi Hanna , Piao Jinhua , Cao Yujia , Hashiguchi Masaaki , Iwai Hideyuki , Amagasa Teruo , Sakaguchi Shimon , Azuma Miyuki
Published: January 2023

We previously reported that GITR acts as a costimulatory molecule in both CD25− CD4+ conventional and CD25+ CD4+ Treg cells using agonistic anti-GITR mAb (DTA-1) (J. Immunol. 2004, 172:7306). However, CD4 T cell responses by the binding of natural ligand, GITRL and the physiological importance of GITR-GITRL pathway have not been well understood. In this study, we examined the involvement of GITR-GITRL pathway in T cell-mediated responses. We established P815 transfectants expressing high levels of mouse GITRL (GITRL/P815) and two anti-mouse GITRL mAbs. Proliferative responses and IL-4 and IL-10 production by CD25− CD4+ T cells in the presence of anti-CD3 mAb were markedly enhanced by GITRL-P815. Anti-GITRL mAb completely inhibited the proliferative responses over the levels of untransfected P815. Surprisingly, the addition of anti-GITRL mAb further enhanced IL-10 production by CD25− CD4+ T cells. Unlike stimulation by DTA-1, CD25+ Treg cells failed to show substantial proliferative responses by GITRL-P815. The addition of CD25+ Treg cells inhibited the proliferative responses enhanced by mGITRL-P815 and the remaining responses were completely inhibited by anti-GITRL mAb. Our results suggest that GITRL-P815 induce potent costimulatory signals to CD25− CD4+ T cells, however, the unknown GITRL pathway may be involved in the T-T interaction.
4-1BB engagement costimulates NKT cell activation and enhances airway hyperreactivity and airway inflammation mediated by NKT cells (88.19)
Authors: kang Chang-Yuil , Kim Dong-Hyeon , Chang Woo-Sung , Lee Yun-Suk
Published: January 2023

Multiple studies have demonstrated that 4-1BB (CD137), a member of the TNF receptor superfamily, is expressed in several immune cells, including activated T cells. However, the expression and the potential roles of 4-1BB on CD1d-restricted invariant NKT (iNKT) cell have not fully characterized. In this study, it was demonstrated that 4-1BB on iNKT cells was consititutively expressed, and the expressions was increased by TCR engagement. Also, 4-1BB signaling provided by 3H3, an anti-4-1BB agonistic mAb, promoted iNKT cells activation, resulting in enhanced proliferation and cytokine production of iNKT cells driven by alpha-galactosylcerimide (alpha-GalCer) or anti-CD3 mAb in vitro. Administration of 3H3 further increased IL-4 and IFN-gamma levels in serum induced by alpha-GalCer administration in vivo. When 3H3 and alpha-GalCer were intranasally co-administrated, airway hyperreacitivity and inflammatory cell accumulation were severely aggravated. In conclusion, 4-1BB on iNKT cell functions as a costimulatory molecule, and facilitates the induction of more severe airway hyperreacitivity and inflammatory cell accumulation in NKT cell-mediated airway inflammation.
Different anti-B7-DC monoclonal antibodies show differential functions in immune responses (88.20)
Authors: Ritprajak Patcharee , Hashiguchi Masaaki , Yagita Hideo , Okumura Ko , Azuma Miyuki
Published: January 2023

Both B7-H1 and B7-DC bind to co-inhibitory receptor PD-1. Various murine disease models using anti-B7-H1mAb (MIH5/MIH6) and/or anti-B7-DC mAb (TY25) demonstrated the preferential involvement of B7-H1 in the PD-1-mediated immune regulation. To verify the actual involvement of B7-DC pathway, we here established another new mAb against mouse B7-DC (MIH37) and compared differential properties between MIH37 and TY25.

Binding analyses using flow cytometry demonstrated that MIH37 showed higher binding affinity to B7-DC. In hapten-induced contact hypersensitivity, the treatment with MIH37 at either sensitization or challenge efficiently inhibited the ear swelling, whereas the treatment with TY25 enhanced at the late time point. Consistently, MIH37 treatment inhibited enhanced induction of activation Ags and IFN-γ production, while TY25 treatment enhanced. When blocking anti-PD-1 mAb was co-administrated at sensitization, the inhibitory effect by MIH37 was reversed at the late time point, whereas the enhancing effect by TY25 appeared at the earlier time point. Our results suggest that MIH37 interferes both B7-DC/PD-1 inhibitory and B7-DC/non-PD-1co-stimulatory pathways, whereas TY25 only blocks B7-DC/PD-1 pathway. PD-1-mediated inhibitory pathway and non-PD-1-mediated co-stimulatory pathway may contribute to the different phases of T cell responses.
B7-H3 co-stimulates T-cell immune responses via the B7-H3 receptor ligation (88.21)
Authors: Hashiguchi Masaaki , Kobori Hiroko , Ritprajak Patcharee , Piao Jinhua , Azuma Miyuki
Published: January 2023

B7-H3 is a new member of B7 family molecules and is widely expressed on various types of tissue and tumor cells as well as immune cells, such as macrophages and dendritic cells. It was suggested that B7-H3 binds a putative counter-receptor on activated T cells that is distinct from the already identified CD28 family molecules. Here we report the functional roles of B7-H3 and its newly identified B7-H3 receptor (B7-H3R) in immune responses.

Co-stimulation of mouse B7-H3 in the presence of CD3 ligation resulted in enhanced proliferation and IFN-γ production by CD8+ T cells in vitro. The in vivo expriments also showed costimulatory roles of B7-H3; Blockade of B7-H3 using anti-B7-H3 mAb inhibited hapten-induced contact hypersensitivity reactions and anti-tumor host responses. Inoculation of B7-H3-introduced tumors efficiently suppressed tumor growth via CD8+ T cell-mediated manners.

We picked up several candidates for B7-H3R from database homology search and identified a B7-H3R that binds to the B7-H3-Fc chimera protein. The transduction of B7-H3R into activated CD4+ T cells and DO11.10 hybridoma enhanced production of IFN-γ and IL-2, respectively. Furthermore, the expression of B7-H3R was recognized in freshly isolated CD8+ T cells and myeloid cells as well as activated CD4+ T cells.

Our results suggested that B7-H3 positively co-stimulates T (preferentially CD8+ T) cell responses via the B7-H3R ligation.
Involvement of GITRL-GITR costimulatory pathway in T cell-mediated immune responses in vivo (88.23)
Authors: Piao Jinhua , Yosuke Kamimura , Hanna Igarashi , Iwai Hideyuki , Massaaki Hashiguchi , Shimon Sakaguchi , Azuma Miyuki
Published: January 2023

GITR is expressed on CD25+ regulatory T cells at high levels. GITR is also expressed on conventional CD4 and CD8 T cells and acts as a potent costimulatory molecule based on the studies using agonistic anti-GITR mAb. The physiological impact of GITR-mediated signal that is delivered by GITRL is still unknown. Here, we report the effects of neutralizing anti-GITRL mAb (MIH44) treatment in tumor and hapten-induced contact hypersensitivity (CH) models.

Inoculation of GITRL-transduced tumor cells (GITRL-P815 and GITRL-SCCVII) into the syngeneic mice efficiently inhibited their tumor growth, suggesting the enhanced antitumor immune responses by tumor-associated GITRL. Administration of anti-GITRL mAb reversed the GITRL-induced antitumor responses, but these effects were decreased in the anti-CD25 mAb treated mice. Treatment with anti-GITRL mAb did not affect the tumor growth of parental tumors.

In a CH model, the anti-GITRL mAb treatment at the sensitization, but not at the challenge, efficiently suppressed ear swelling. This inhibitory effect was long-lasting. The anti-GITRL mAb treatment significantly decreased the percentages of IFN-γ+CD8+ and CD25+ T cells. These inhibitory effects by anti-GITRL treatment were reduced in the anti-CD25 pre-treated mice.

Our data suggest that GITR-GITRL costimulatory pathway is involved certainly in activation of conventional T cells and regulatory T cells may affect the GITRL-costimulatory pathway.
Foxp3+ Treg Cell Activity increases in Secondary Lymphoid Organs of Aged Mice (88.25)
Authors: Chiu Bochin , Stolberg Valerie , Chensue Stephen
Published: January 2023

As potent suppressors of immune responses to self and foreign antigens, Foxp3+ Treg cells are suspected to be involved in immunosuppression leading to cancer, neurodegeneration and infection. Since aging is associated with increased incidence of these diseases, we compared Treg activity in blood, lymphoid organs and lungs of young (5–6 months) and old (21–22 months) C57BL/6 mice, using flow cytometric detection and characterization of Foxp3+ cells. Both the proportion and absolute number of Treg cells increased with age in secondary lymphoid organs but not in blood and lungs. Whereas numbers of thymic and peripheral naive Treg cells decreased with age, the number and proportion of Treg cells with CD103+ memory/effector phenotype increased in peripheral lymphoid tissues. Moreover, the increased Treg activity had in vivo influence since CD40 and CD86 co-stimulatory molecule expression by lymph node dendritic cells (DCs) was impaired in old mice, but could be restored to levels of young mice by inactivating Treg cells with anti-CD25 mAb. Our findings suggest that immunosenecence involves a cumulative imbalance of Treg cell activity and have profound implication for the understanding of age-associated immune dysfunction.

This work was supported by the American Foundation for Aging Research (AFAR) and the Department of Veterans Affairs.
The inverse agonist activity of soluble CTLA-4 ligands requires oligomerization of this receptor and CD28 expression (88.26)
Authors: Teft Wendy A , Madrenas Joaquin
Published: January 2023

CTLA-4 can inhibit or activate T cells depending on the ligand it engages. We have shown that CTLA-4 ligation with a bi-specific in-tandem single chain Fv, known as 24:26, can activate CTLA-4-expressing primary human T cells in the absence of TCR and CD28 engagement. To understand the basis of such an inverse agonist activity, we examined the requirements for CTLA-4-mediated T cell activation. We observed that 24:26 and soluble B7.1 Ig, but not B7 molecules on APCs, induced the formation of a unique CTLA-4 oligomer that disappeared from the soluble cell lysate fraction and subsequently appeared in the insoluble cellular fraction. Oligomerization occurred upon ligation of CTLA-4 homodimers and was necessary but not sufficient for T cell activation. The inverse agonist activity of these soluble ligands required the cytoplasmic domain of CTLA-4 since CTLA-4 molecules lacking this domain did not induce T cell activation. CD28 expression was necessary for the inhibitory and activating functions of CTLA-4 suggesting a functional interaction between these two receptors.

Understanding the functional responses to soluble ligands of costimulatory receptors is important in the wake of the side effects observed with CD28 superagonists. Our data indicates that the biological effect of soluble ligands of CTLA-4 will be determined by the oligomerization of this receptor.

Supported by the Canadian Institutes of Health Research.
T Cells are Capable of Effector and Regulatory Function Simultaneously during Activation (88.3)
Authors: pillai vinodh
Published: January 2023

CD4+CD25+ regulatory T (Treg) cells are an important arm of the immune system that downregulate potentially harmful effector immune responses. FOXP3 expression and in vitro suppressor function have been considered to be the hallmarks of a unique and stable lineage of T-cells with immune regulatory function. Here we show that human CD4+CD25−FOXP3− T-cells attain a transient FOXP3+CD25+ state following activation. In this state of activation, these cells possess the classic marker profile and suppressive phenotype of regulatory/suppressor T-cells. However, the vast majority of these cells eventually down regulate FOXP3 and suppressor activity. Cytokine studies indicate that they are also capable of secreting effector cytokines when they are regulatory in function. Interestingly, CD8+CD25−FOXP3− T-cells also exhibit similar dynamics following activation. Our results show that, in humans, FOXP3 expression and Treg functionality are not distinct features of a separate lineage of T-cells, but rather a state attained by almost all T-cells during their activation. This suggests that attempts to increase “Tregs” in humans may paradoxically result in increased effector T-cells, with potentially deleterious effects. These results warrant caution in interpreting human studies using FOXP3 and suppressive activity as readouts.

This work was supported by grants (to N.J.K) from the NIH and the National MS society NMSS.
CD4 T cell expression of costimulatory receptors is directly enhanced by Toll-like receptor ligands (88.37)
Authors: Taylor Devon K , LaRosa David F , Rahman Adeeb H , zhang Jidong , Turka Laurence A
Published: January 2023

Toll-like receptors (TLR) play an integral role in bridging the innate and adaptive response. The stimulation of TLRs on antigen presenting cells promotes their maturation through the upregulation of MHC class II molecules, costimulatory ligands, the release of pro-inflammatory cytokines.

Emerging data support the presence of TLR2, 3, 5, 8 and 9 on non-regulatory CD4+ T cells. Our group has shown the direct ligation of TLR 9 on activated CD4+ T cells promotes survival and proliferation through the production of IL-2 and the activation of PI3K and NF-κB-dependent pathways.

Using mice deficient in MyD88, TLR9 and TLR3, we investigated the effects of TLR ligation on T-cell costimulatory receptor expression. We found that stimulation of activated CD4+ T cells with TLR ligands leads to the selective upregulation of costimulatory receptors in both a MyD88 and TLR9 dependent manner. Both CpG DNA and dsRNA synthetic analog poly(I:C) enhanced the expression of OX40, GITR, CTLA-4 and CD28 in activated CD4+ T cells. Furthermore, responses to CpG DNA were TLR 9 and MyD88 dependent, although responses to poly(I:C) were not dependent on TLR3. Ongoing studies examine the functional consequences of TLR-enhanced costimulatory receptor expression in T cells and focus on its contribution to effector function and memory cell formation.

This work is funded by NIH grant (R01-AI-062789) to L.A.T.
Mechanism of Action of Abatacept: Insights from Predictive Biosimulation Studies (88.38)
Authors: Whiting Chan Chung , Ramanujan Saroja , Young Daniel L. , Kellermann Sirid-Aimee , Defranoux Nadine A.
Published: January 2023

While clinical reports demonstrate that CTLA-4 Ig (abatacept) is efficacious in rheumatoid arthritis (RA) patients refractory to standard therapies, its mode of action is poorly characterized. Using the Entelos® RA PhysioLab® platform, a mechanistic mathematical model of a prototypical human RA joint, we evaluated abatacept’s potential mechanisms of action to gain insight into the basis of its clinical efficacy. The hypothesized effects of abatacept on (a) T cell functions (e.g., inhibition of proliferation, costimulation, and Th1 activity and induction of T regulatory cells (Tregs)), (b) antigen presenting cell functions (e.g., inhibition of cytokine production and antigen presentation), and (c) cross talk between T cells and macrophages, fibroblast-like synoviocytes, and dendritic cells were investigated by altering the model values that define each process. Biosimulation of abatacept therapy demonstrated that a 50% modulation of these targeted functions is sufficient to reproduce clinical responses in a ‘virtual’ RA patient, whereas a 20% effect is insufficient. Analysis of these results shows that inhibition of macrophage activation and cytokine production, and skewing of T cells to a Treg phenotype drive abatacept efficacy in silico. By reducing the uncertainty regarding the mechanisms critical for abatacept efficacy, biosimulation can lead to better and more targeted RA treatments.
CD25+CD4+ T regulatory cells utilize β-galactoside-binding protein to suppress proliferation of CD8+T cells. (88.40)
Authors: Baatar Dolgor , Olkhanud Purevdorj , Almanzar Giovanni , Wells Valerie , Mallucci Livio , Biragyn Arya
Published: January 2023

CD25+CD4+ T regulatory cells (Tregs) are important cells that control induction of autoimmunity. They induce peripheral T cell tolerance via contact-dependent process, though the precise mechanism remains poorly understood. Here we report that βGBP (β-galactoside binding protein), a cytokine with a physiological role in cell cycle control and a putative role in the silencing phase of T cell immune response and the switching off of T cell effector functions, participates in Tregs-mediated regulation of T cells. We found that freshly isolated human PBL-derived natural Tregs constitutively express βGBP and utilize it to mediate suppression of proliferation of naïve CD8+ T cells. Although large amounts of βGBP can be secreted from stimulated Tregs, suppression of proliferation by βGBP is mediated by nM concentrations of βGBP bound to the cell surface, which is in accord with its cytokine-like function. Interestingly, Tregs utilize βGBP to specifically and transiently inhibit proliferation of CD8+ T cells without inducing apoptosis. Inhibition of proliferation is associated with the ability of βGBP to inhibit downstream signaling events of TCR. Our data indicate that Tregs use βGBP as a key mediator of contact-dependent regulation of T cell proliferation.

This work was supported by the Intramural Research Program of the National Institute on Aging, NIH.
Injury- and antigen-specific control of CD4 T cell reactivity by CD4+CD25+ regulatory T cells (88.42)
Authors: MacConmara Malcolm , Delisle Adam , Stallwood Christopher , McKenna Ann , Mannick John , Lederer James
Published: January 2023

Severe injury augments the potency of CD4+CD25+ regulatory T cells (Tregs) suggesting that Tregs might play a role in controlling unwanted CD4 T cell responses in the injured host. To test whether enhanced Treg activity might occur by an antigen driven process, we compared T cell responses in Treg-depleted sham and burn mice that received unsorted or CD25-depleted CD4+ T-cells from DO-11.10 T cell receptor (TCR) transgenic mouse. Unsorted DO-11.10 CD4+ T cells contain 10% CD25+FoxP3+ T-cells. Treg-depleted or control mice received 5 x 106 CD4 T cells or CD25-depleted CD4 T cells by i.v. injection, were immunized OVA323-339 peptide and then underwent sham or burn injury under anesthesia. At day 7, lymph node and spleen cells were prepared and stained with DO-11.10 TCR specific antibody. We observed a significant increase in DO-11.10 T cell expansion in sham and burn Treg-depleted mice that received CD4+CD25− T cells. This increased expansion occurred in sham, but not burn mice, receiving unsorted CD4 T cells suggesting that the co-transfer of CD25+ DO-11.10 T cells was sufficient to suppress T cell expansion in injured mice. The results of CFSE-studies confirmed that antigen-stimulated CD4+CD25− T-cells proliferate more vigorously in burn as compared to sham mice, but that transferring unsorted T cells prevents the injury-induced CD4 T cell expansion. These results indicate that injury influences Treg activity in an antigen-specific fashion and suggest that Tregs may play an active role in modulating CD4 T cell reactivity following injury
PD-1 plays a stimulatory role in CD8 T cell priming and an inhibitory role after activation (88.43)
Authors: Chen Jian , Zajac Allan , Wu Qi , Yang Pingar , Xu Xin , Hsu Hui-Chen , Mountz John D
Published: January 2023

The expression of the program death-1 (PD-1) receptor, a member of the CD28 costimulatory receptor family, is induced by T cell activation. It is still controversial whether PD-1 plays an inhibitory role or act as a stimulatory factor in T cell activation. It is yet to be determined if PD-1 contributes to age-related defect in CTL response to virus. We hypothesized that in the initiation stage, PD-1 plays a stimulatory role in T cell activation, and that upregulated PD-1 on activated T cells delivers an inhibitory signal to T cells. In the present study, we measured PD-1 expression in aged mice after adenovirus (Ad) infection and correlated it with Ad specific CTL activation using an MHC I tetramer, Db-E1Bp. First, to determine the need for PD-1 in the primary CTL response, we administered Ad into PD-1 deficient mice (PD-1 KO) in parallel with CD28 deficient (CD28 KO) mice. There is a significant decrease in the generation of Db-E1Bp+CD8+ cells in both CD28 KO and PD-1 KO mice 8 days after Ad infection, compared to WT mice (1% and 4% of total CD8+ cells, respectively, compared to 9%. p < 0.05). We next demonstrated that PD-1 upregulation was associated with a decrease in Ad specific CTL response and a defect in activation induced cell death in aged mice. Eight days after Ad infection, 83% of Db-E1Bp+CD8+ cells expressed high level of PD-1 in 24-mo old mice, compared to 16% in young mice (p < 0.05). The expression of PD-1 on non-activated, Db-E1Bp−CD8+ cells was similar in young and old mice. These results indicated that PD-1 on naïve CD8 T cells plays a costimulatory role in T cell activation and high level of PD-1 expression induced upon activation turn it into an inhibitory receptor. Its inhibitory function might also account for the defective activation induced cell death in virus specific CD8 T cells in old mice.
Contrasting effects of Cyclosporine and Rapamycin in de novo generation of alloantigen-specific regulatory T cells (88.45)
Authors: Gao Wenda , Lu Yan , ElEssawy Basset , Oukka Mohamed , Kuchroo Vijay , Strom Terry B
Published: January 2023

The outcome of T cell-mediated responses critically depends on the balance of cytopathic versus regulatory T (Treg) cells. Induction of stable tolerance to allografts requires limiting donor-reactive T effector (Teff) cells via apoptosis. Cyclosporine (CsA) blocks AICD of Teff cells, and is detrimental to co-stimulation blockade-induced tolerance, whereas Rapamycin (RPM) preserves AICD, potentiating such tolerance. While differences between CsA and RPM in influencing apoptosis of Teff are apparent, their effects on graft-protective Treg cells remain enigmatic. Moreover, it is unclear whether tolerizing regimens foster conversion of naïve peripheral T cells into alloantigen-specific Treg cells. Here we show, using reporter mice for Treg marker Foxp3, that RPM promotes de novo conversion of alloantigen-specific Treg cells, whereas CsA completely inhibits this process. Upon transfer, in vivo converted Treg cells potently suppress the rejection of donor but not third party skin grafts. Thus, the use of RPM over CsA is favorable in shifting the balance of aggressive to protective type alloimmunity.

(Supported in part by grants from NIH and JDRF (NIH NS 30843; NIH A141521-08; JDRF 5-2005-108)).
Reciprocal generation of Treg and Th1/Th17 cells by conventional B cells and B1 B cells (88.46)
Authors: Zhong Xuemei , Gao Wenda , Degauque Nicolas , Bai Chunyan , Kenny James , Lu Yan , Oukka Mohamed , Strom Terry B , Rothstein Thomas L
Published: January 2023

Regulatory T cells (Treg) are indispensable for maintaining peripheral tolerance, whereas Th1 and Th17 cells induce inflammation and tissue destruction. Here we report a novel regulatory role for B cell subsets in influencing the differentiation of Treg versus Th1/Th17 cells. Using Foxp3-GFP knock-in mouse, we demonstrated that conventional B cells excelled in converting Foxp3− CD4 T cells into Foxp3+ Treg cells in the presence of IL-2 and TGFβ, whereas under the same conditions, peritoneal B1 cells preferentially generated Th1 and Th17 cells. We also demonstrated that B1 B cells promote stronger T cell proliferation during antigen presentation and MLR as compared to conventional B cells. Costimulation blockade of CD86 but not CD80 partially reversed the poor Treg conversion by B1 cells. Hence the ability to convert Treg is inversely correlated with B cell antigen-presentation capacity. Our findings suggest that different B cell subsets can play distinct roles in immune regulation by polarizing T cell functions.

This study was supported by grants from the American Heart Association (0325807T), the Juvenile Diabetes Research Foundation (5-2006-19) and the National Institutes of Health (AI29690 and AI60896).
Crystal structure of SLAM family members: implications for their costimulatory functions in T cell (88.9)
Authors: Yan Qingrong , Malashkevich Vladimir , Cao Erhu , Lary Jeffrey W , Cole James L , Nathenson Stanley G , Almo Steven C
Published: January 2023

Members of the signaling lymphocyte activation molecule (SLAM) family, including homophilic and heterophilic receptors, modulate a wide range of immune responses, including T cell activation and NK-cell activation. The SLAM family members share a similar ectodomain organization, with an N-terminal IgV domain and a C-terminal IgC2 domain. The sole exception is the Ly-9 ectodomain, which is composed of a tandem repeat of the IgV-IgC2 motif. The homophilic interaction is mediated by the first N-terminal IgV domain. The 2.0 Å crystal structures of the CD84 and Ly9 first N-terminal IgV domains reveal a similarly organized homophilic interaction with the recently reported NTB-A homophilic interaction, the differences in the homophilic interfaces is likely an important mechanistic feature that prevents the formation of undesired heterodimers among the SLAM family receptors. Notably our biochemical data also indicated the strong self association of CD84 and Ly9 with a Kd in the sub-micromolar range. The fact that the homophilic affinities span over three orders of magnitude among the SLAM family members suggests that the difference in the affinities might be a contributor to the different signaling behaviour exhibited by the individual family members.
In Vitro Human Immune Response to Low Dose Mercury (89.11)
Authors: Gardner Renee Michelle , Nyland Jennifer F. , Evans Sean , Silbergeld Ellen K.
Published: January 2023

There is growing recognition that exposures to both inorganic and organic mercury (Hg) compounds can affect immune function, with potential consequences for host resistance to infections and risks of autoimmune disease. However, there is little information on human responses to low level Hg exposures. For this reason, we are investigating immune function using in vitro exposures to Hg in primary cultures of human peripheral blood mononuclear cells (PBMCs) from healthy adult volunteers. PBMCs were exposed to a concentration of HgCl2 up to 200 nM (which roughly corresponds to a whole blood concentration at the upper range of occupationally exposed or fish eating populations), in the presence and absence of activation by LPS. We found that these concentrations are non-cytotoxic and do not affect cell subsets or markers of cell activation. However, in PBMCs from both males and females, dose-dependent increases in both TNFα and IL-6 were observed in cell culture supernatants in the presence of LPS. No changes were observed in IFNγ, IL-4, IL-5, or IL-13 levels. No changes were seen in any cytokine in the absence of LPS. The results indicate that Hg can exert significant effects on signal transduction pathways in human cells in vitro, at exposures that are non-cytotoxic and environmentally relevant.