bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytokine treatment of macrophage suppression of the Mls response (88.3)
Authors: Swider Adam , Kozlowski Megan , Silberman Daniel , Bucknum Amanda , Matlack Robin , Orlowski Michelle , Riggs James
Published: January 2023

Components of the immune system essential for curbing tumor progression are often found within the tumor. High macrophage to T cell ratios (Mö:T) are hallmarks of the tumor microenvironment. We have shown that such conditions suppress the immune response to the retroviral superantigen Mls by IFN ã -triggered production of the Arg- and Trp-consuming enzymes iNOS and IDO. In attempts to reverse suppression by treatment with pro-inflammatory cytokines, IL-6 improved the T cell response to Mls, IL-1 and TNFá had little effect, and the pro-hematopoietic cyokines IL-3 and GM-CSF increased suppression. GM-CSF treatment increased Mö expression of CD80, a significant ligand for the immunosuppressive B7H1 and CTLA-4 receptors. These results reveal avenues for reversal of the suppression of cell-mediated immunity characteristic of the high Mö:T cell ratios found in many tumors. Supported by NIH AREA R15-AI060356-01.
Induction of peripheral Tregs by homotypic T cell interaction (89.3)
Authors: Thuemmler Katja , Ramming Andreas , Leipe Jan , Schulze-Koops Hendrik , Skapenko Alla
Published: January 2023

The mechanisms by which regulatory T cells are generated in the periphery are incompletely understood. Here we show that homotypic T cell interaction results in the generation of T cells with a potent regulatory capacity.

CD4 memory T cells that were cultured together with activated effector T cells proliferated and produced various cytokines depending on the nature of the activated effector T cell. Whereas Th1 cells promoted the development of IL-10 and/or IFNγ producing cells, T cells cultured with Th2 cells produced IL-4. The interaction between the adhesion receptors ICAM-1, 2, 3 and LFA-1, respectively was crucial for T-T cell communication with regard to proliferation and cytokine production. Blocking of ICAM-1 diminished IFNγ production, whereas ICAM-3 was important for IL-4 secretion. Interestingly, the resulting cells strongly inhibited proliferation of CD25- T cells in a dose dependent manner independent of their cytokine secretion profile. Moreover, in vitro generated T-T cell induced T cells with a TCR specific for OVA prevented the expansion of OVA specific effector T cells in Balb/c mice in vivo upon antigen challenge.

The results indicate that homotypic T cell interaction induces the generation of T cells with a regulatory phenotype, providing a novel negative feedback mechanism to control sustained T cell driven immunity.
Induction of granzyme B expression in human regulatory T cells requires TCR and CD28 triggering but not IL-2 and is suppressed by inhibitors of the PI3K-mTOR pathway. (89.40)
Authors: Kelley Todd W. , Efimova Olga
Published: January 2023

Regulatory T cells (Tregs) can employ a cell contact- and granzyme B-dependent mechanism to mediate suppression of bystander T and B cells. Granzyme B may also be involved in the suppression of anti-tumor immunity in the tumor microenvironment. In spite of its central importance, a detailed study of granzyme B expression patterns in Tregs has not been performed. Using enriched cultures of CD4+,CD25+,FOXP3+ T cells freshly isolated from the peripheral blood of normal adults, we demonstrate that natural Tregs lack granzyme B expression. Tregs subjected to prolonged TCR and CD28 triggering express high levels of granzyme B but CD3 stimulation alone fails to significantly induce such a response. IL-2 stimulation alone also fails to induce granzyme B and there is no co-requirement for exogenous or endogenous IL-2 for granzyme B expression. We find that treatment of Tregs with the mTOR inhibitor rapamycin or the PI3 kinase (PI3K) inhibitor LY294002 markedly suppresses granzyme B expression. However, neither rapamycin, as previously reported by others, nor LY294002 inhibits Treg proliferation or induces significant cell death in TCR/CD28/IL-2 stimulated cells. Rapamycin, but not LY294002, selectively favors the expansion of Tregs over conventional CD4+ T cells. Thus, we conclude that TCR/CD28 mediated activation of mTOR is necessary for granyzme B expression but not proliferation in regulatory T cells. These findings may have implications for the biology of regulatory T cells generated in the clinical setting of rapamycin treatment.
The Effect of Phosphatidylserine on T Cell and Dendritic Cell Function (90.2)
Authors: Xia Changqing , Gangrade Abhishek , Peng Ruihua , Clare-Salzler Michael J
Published: January 2023

Phosphatidylserine (PS) is a phospholipid of plasma membrane. PS is located in the inner leaflet of plasma membrane but turns to outer leaflet when cell death program is initiated. It has been demonstrated that PS on apoptotic cells plays an important role in modulating immune responses. However, whether apoptotic cells release soluble PS which may subsequently affect immunocytes is not known. Recently, we found that apoptotic cells released soluble PS to the culture media which could block the binding of anti-PS antibody to apoptotic cells. To address how soluble PS influences T cell and dendritic cell function, we examined the effect of water-soluble PS (Avanti Polar Lipids, >95% purity with no endotoxin) on T cell proliferation, and monocyte-derived dendritic cell (DC) cytokine and chemokine production. Our results demonstrated that PS suppressed T cell proliferation in a significant dose-dependent manner. However, PS appeared not affecting T cell early activation based on the normal up-regulation of CD69. On DCs, PS did not induce any cytokine or chemokine production. However, PS significantly altered LPS-stimulated DC cytokine and chemokine producing profiles. We found that PS enhanced LPS-induced IL-1beta, IL-alpha, GM-CSF and IL-6, but suppressed IL-10, IL-12p40 as well as most chemokines tested. There was no significant influence on DC maturation. Our findings indicate that the apoptotic cells may modulate immune responses through releasing soluble PS. The mechanisms of PS effect on T cells and DCs are being investigated.
Thy-1 signaling in murine T cells promotes cytokine polarization (90.20)
Authors: Furlong Suzanne J , Hoskin David W
Published: January 2023

Glycosylphosphatidylinositol-anchored proteins (GPI-AP) that provide activating signals to T cells have been implicated in T cell-mediated immune responses. The effect of GPI-AP signaling on cytokine production and the development of effector T lymphocyte phenotypes remains unclear. In this study, highly pure resting T cells were stimulated with either anti-Thy-1 or anti-TcR monoclonal antibody (mAb) along with costimulatory signals provided by LPS-matured dendritic cells. At concentrations of mAb that induced optimal proliferation, Thy-1-activated T cells proliferated ~2-fold less than TcR-activated T cells. Analysis of cytokine production using cytokine arrays and ELISA showed that Thy-1 signaling induced significantly less IL-2 and IFN-γ synthesis than TcR signaling. Interestingly, Thy-1-stimulated T cells produced significantly more IL-17 and IL-4 than TcR-stimulated T cells. Cytokine polarization was unique to Thy-1, as T cells stimulated with mAb against the GPI-AP Ly6A/E did not produce appreciable levels of IL-17 or IL-4. These results support previous reports that Thy-1 signaling differs from TcR signaling and suggest that Thy-1 may provide polarizing signals that promote the development of Th17 and Th2 cells. Thy-1 and other GPI-AP proteins require further study to elucidate their possible roles in influencing the nature of T cell-mediated immune responses. Research was funded by NSERC
Combining PDL-1 blockade with agonistic antibodies against 4-1BB enhances T cell restoration after chronic LCMV infection (90.22)
Authors: Penaloza MacMaster Pablo , Vezys Vaiva , Ha Sang-Jun , Konieczny Bogumila , Barber Daniel , Mittler Robert , Ahmed Rafi
Published: January 2023

The PDL-1:PD-1 pathway is involved in T cell exhaustion after chronic viral infection. PD-1 can bind to either PDL-1 or PDL-2, whereas PDL-1 can bind to either PD-1 or B7.1. We tested the effect of combining standard aPDL-1 blockade with a4-1BB (3H3 agonistic antibody) treatment to determine T cell restoration effects in a model of chronic infection with lymphocytic choriomeningitis virus (LCMV). We chose 4-1BB because it has been shown to be an important costimulatory pathway. It appears that a4-1BB treatment enhances the effect of aPDL-1 blockade if given in a low dose (50 μg, once). Conversely, if a4-1BB is given in a high dose (200 μg, five times), it results in sudden increase of antigen-specific T cells by day 7, but massive T cell death by day 14 post treatment. These results show the importance for 4-1BB costimulation in aPDL-1 mediated T cell restoration.
Selective induction of robust antibody responses following antigen targeting to dendritic cell subsets (90.34)
Authors: Chappell Craig P , Draves Kevin E , Clark Edward
Published: January 2023

Dendritic cells (DCs) are essential for the initiation and regulation of immune responses. CD8α+ DCs in the mouse spleen express DEC-205 (CD205), while many splenic CD8α- DCs express DC-inhibitory receptor-2 (DCIR2/33D1). Delivering antigen (Ag) via mAbs to either DEC-205 or DCIR2 was shown by other groups to preferentially induce CD8+ and CD4+ T cell responses, respectively. However, the ability of these two DC subsets to induce primary Ab responses is not well defined. Using the conventional hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) coupled to mAbs that recognize DEC-205 or DCIR2, we demonstrate that DCIR2+ DCs, but not DEC-205+ DCs, are able to induce robust anti-NP Ab responses following intravenous injection of soluble Ag. These Ab responses were restricted to the IgG1 subclass with little to no IgM or IgG2a production. Surprisingly, the DCIR2-induced Ab response did not require the addition of conventional adjuvants. These responses were both TCR and CD40-dependent, indicating that T-B cell collaboration is required. Finally, we found that CD22 expression by B cells was also required for IgG1 anti-NP responses, suggesting that DC-B cell interactions may be important during early B cell priming via this route of administration. Together these results demonstrate that, similar to the selective priming of T cells by these DC subsets, DCs are also specially equipped to selectively prime B cells and Ab responses. This project was supported in part by NAIAD training grant T32AI007411 and by NIH grant AI52203 and does not necessarily represent the official views of the NAIAD or the NIH.
Differential effects of negative costimulatory molecules BTNL2 and PD-L1 on the activation of T cells when using MHC class I tetramer, MHC class II tetramer, or anti-CD3 immobilized on beads (90.6)
Authors: Swanson Ryan , Viney Joanne L
Published: January 2023

Immunologists have identified a growing list of negative costimulatory molecules in the B7 family of proteins. These molecules are defined in vitro by their capacity to inhibit purified T cell proliferation and cytokine production only when immobilized proximally to anti-CD3. We are interested in whether the negative costimulatory molecules behave differently when activating T cells in an antigen specific manner. The required proximal immobilization with TCR agonist can be achieved on beads loaded with MHC class I or class II tetramers and subsequent culture with appropriate transgenic T cells. In this poster, we show evidence that CD4 and CD8 T cells are inhibited differently when stimulated with peptide/MHC molecules compared to activation with anti-CD3. The two negative costimulatory molecules we are currently studying, BTNL2 and PDL1, block activation under different circumstances. Although both molecules can inhibit T cell responses to anti-CD3, PDL1 is more effective at inhibiting CD8 responses to MHC class I than CD4 responses to MHC class II. Conversely, BTNL2 inhibits CD4 T cells in the same system, but cannot block the CD8 response as well as PDL1. These studies imply that different negative costimulatory molecules may have cell specific roles and provide a system to investigate regulatory mechanisms that result in differential inhibition of CD8 and CD4 T cells.
IL-15 produced and trans-presented by DCs underlies homeostatic competition between CD8 and gd T cells under lymphopenic conditions (90.7)
Authors: Do Jeong-su , Min Booki
Published: January 2023

Naive T cells undergo proliferation in lymphopenic environments. However, cellular mechanisms controlling the proliferation and the maintenance of T cells under these conditions remain elusive. Here, we compared naive T cell proliferation and homeostatic maintenance under different lymphopenic conditions: TCRβ-/- and Rag1-/-. Both CD4 and CD8 T cells fully diluted CFSE content under these conditions. While CD4 and CD8 T cells equally proliferated in Rag1-/- mice, enhanced CD4 and impaired CD8 T cell expansion was found in TCRβ-/- mice. B cells played little role in the differential expansion of T cell subsets. However, depletion of γδ T cells in TCRβ-/- mice was sufficient to restore the impaired expansion of CD8 T cells. Similarly, adoptive transfer of γδ T cells into Rag1-/- recipients altered expansion of T cell subsets similar to that within TCRβ-/- recipients. DCs in Rag1-/- mice expressed higher levels of IL-15Rα as well as of membrane bound IL-15 than DCs in TCRβ-/- mice. Adoptive transfer of wild type DCs into TCRβ-/- recipients enhanced CD8 T cell expansion, yet such enhancement was absent following IL-15-/- DC transfer. Collectively, our findings demonstrate that γδT cells suppress naive CD8 but not CD4 T cell expansion under lymphopenic conditions by competing IL-15 trans-presented by DCs, revealing previously unrecognized homeostatic mechanisms between different T cell subsets in vivo.
Menopause accelerates immune senescence and diminishes immune response to vaccination (90.8)
Authors: Messaoudi Ilhem , Engelmann Flora , Barron Alexander , Urbanski Henryk , Neuringer Martha
Published: January 2023

Menopause and the associated loss of estrogen and progesterone represent the most dramatic physiological change in aging women. Aging is accompanied by a decline in immunity referred to as immune senescence that significantly increases morbidity and mortality in older individuals. Several studies strongly suggest that female sex hormones influence the immune system. However, the impact of menopause on immune senescence remains poorly understood. To address this question, menopause was induced surgically in adult and aged female rhesus macaques by bilateral oophorectomy. Our studies show that ovariectomized adult female rhesus macaques generate a lower antibody and T cell responses following vaccination than age-matched cycling females. Furthermore, ovariectomized female rhesus macaques have increased frequency of CD8CD28- T cells, which suggest accelerated T cell senescence. Estrogen replacement therapy (ERT) improved both T and B cell responses to vaccination in elderly oopherectomized female rhesus macaques whereas combined hormone replacement therapy had modest effects. These data suggest that ovarian senescence exacerbates immune senescence and that ERT can improve immune response to vaccination in aged postmenopausal females.
Cysteine Protease Activators of Plasma Chemoattractant Chemerin (92.4)
Authors: Cichy Joanna , Kulig Paulina , Stefanska Anna , Kantyka Tomasz , Zabel Brian A , Potempa Jan , Butcher Eugene C
Published: January 2023

Chemerin, a ligand for seven-pass transmembrane, G protein-associated receptor CMKLR1 requires C-terminal proteolytic processing to unleash its chemoattractant activity. Proteolytically-processed chemerin selectively attracts specific subsets of immunoregulatory antigen presenting cells, such as immature plasmacytoid dendritic cells (pDC) and macrophages that express CMKLR1. Chemerin is predicted to belong to the structural cathelicidin/cystatin family of proteins that comprise inhibitors of cysteine proteinases (cystatins). Cysteine proteinases of the papain-like family are well characterized proteolytic enzymes, best known for their role in intracellular proteolytic pathways. Nevertheless, recent studies show that lysosomal cathepsins can also exert their proteolytic activity at extracellular sites in secreted or cell surface-bound forms. Extracellular cathepsins contribute to a variety of pathophysiological processes, including inflammation and neoplastic progression. Homology between chemerin and cystatins suggests that chemerin interacts directly with cysteine proteases. In this work, we demonstrate that chemerin does not display substantial inhibitory activity against human cysteine proteases. Instead, chemerin was found to be a new substrate for these proteases. The interaction between chemerin and the proteases resulted in the generation of potent attractant activity, eliciting a robust chemotactic response by CMKLR1 transfectants as well as human blood-derived PDC. Our data demonstrate that cathepsins may contribute to immune responses through the recruitment of pDC.
Severe impairment of leukocyte recruitment into inflamed tissue of ppGalNAcT1-deficient mice (94.1)
Authors: Mueller Helena , Tenno Mari , Van Aken Hugo , Marth Jamey D. , Ley Klaus , Zarbock Alexander
Published: January 2023

P-selectin glycoprotein ligand-1 (PSGL-1) plays an important role in leukocyte recruitment. Its binding affinity to selectins is modulated by posttranslational modifications. The polypeptide N-acetylgalactosamine transferase-1 (ppGalNAcT1) initiates core-type protein O-glycosylation. To address whether the glycosylation of PSGL-1 by ppGalNAcT1 is important for leukocyte recruitment, we investigated leukocyte rolling and velocity in untreated and TNF-α treated cremaster muscles and autoperfused flow chambers comparing ppGalNAcT1-/-(Galnt1-/-) with WT mice. In untreated and TNF-α treated Galnt1-/- mice, leukocyte rolling was significantly reduced with markedly increased rolling velocity compared to control mice. Flow chamber experiments showed that Galnt1-/-- and WT-neutrophils had the same rolling velocity on E-selectin, but the rolling velocity of Galnt1-/--neutrophils on E-selectin/ICAM-1 was significantly elevated, suggesting that E-selectin-dependent neutrophil activation may be defective. Thioglycollate-induced peritonitis experiments with chimeric mice revealed that hematopoietic ppGalNAcT1 is important for leukocyte recruitment. These data show that the loss of ppGalNAcT1 led to reduced leukocyte rolling and recruitment and increased rolling velocity, suggesting a predominant role of ppGalNAcT1 in attaching functionally relevant O-linked glycans to PSGL-1.
LOX-1 is a novel mediator of acute lung inflammation (94.10)
Authors: Fu Jian , Cheng Lili , Liang Mei , Zhang Ping
Published: January 2023

Up-regulation of Lectin-like oxidized LDL receptor-1 (LOX-1) has been known to mediate oxidized LDL-induced vascular inflammation and atherogenesis. Even though there has been increasing evidence suggesting that LOX-1 may play an important role in chronic inflammation, the role of LOX-1 in acute inflammation and tissue injury remains largely unknown. In our present study, we assessed the role of LOX-1 in a mouse model of endotoxin-induced acute lung injury. We demonstrated that LOX-1 could act directly as a neutrophil adhesion molecule. Importantly, blockade of LOX-1 was able to inhibit neutrophil infiltration into the lung. Furthermore, LOX-1 appears to play an important role in mediating pro-inflammatory signaling. NF-κB activation, ICAM-1 expression and apoptotic signaling induced by endotoxin challenge in the lung were all significantly inhibited by LOX-1 blockade. Collectively, our results indicate that LOX-1 could be a multi-functional pro-inflammatory mediator that may play important role in both the induction and development of acute lung inflammation and injury. Our studies also suggest that LOX-1 may serve as a potential therapeutic target against septic shock.
CD98hc Enables B cell Clonal Expansion and Adaptive Humoral Immunity (94.11)
Authors: Cantor Joseph , Browne Cecille , Feral Chloe , Rickert Robert , Ginsberg Mark
Published: January 2023

Lymphocyte clonal expansion mediates adaptive immunity. Here we report that CD98hc enables humoral immunity by supporting B cell clonal expansion. Deletion of B cell CD98hc in mice through conditional gene targeting did not affect B cell compartmentalization, but did result in reduced antibody responses. The mechanism of this effect did not involve interference with early signaling from antigen receptors, but instead was due to total suppression of rapid B cell proliferation, and consequent inability to form plasma cells. CD98hc can mediate both integrin signaling and amino acid transport. Reconstitution with CD98hc mutants revealed that its integrin interaction is required and its amino acid transport function is dispensable for B cell proliferation. These data establish that CD98hc enables clonal expansion of B cells, show that this function is performed by support of cell proliferation through interaction with integrins, and suggest that adaptive immunity drove appearance of CD98hc and its ability to capacitate integrin signaling.
T cell trans-endothelial migration and homing to lymph nodes rely on Myosin-IIA mediated acto-myosin contractility (94.25)
Authors: Jacobelli Jordan , Friedman Rachel S. , Conti Mary Anne , Khan Omar , Sorensen Caitlin , Adelstein Robert S. , Krummel Matthew F.
Published: January 2023

T lymphocytes migrate from the vasculature into lymph nodes and tissues scanning for antigenic peptides on cell surfaces. How T cells migrate through different environments and switch between types of motility in response to different stimuli is not fully understood. We have previously shown that in vitro Myosin-IIA has a role in T cell migration by regulating the switch between fast amoeboid-like motility and slower mesenchymal-like motility. Here, using conditional knockout mice, we have studied the role of Myosin-IIA-dependent cytoskeletal contractility in the migration of primary T cells in vivo. While Myosin-IIA depleted T cells in the lymph node were motile, they did not achieve the same fast motility rates of control T cells. In addition, homing of T cells to lymph nodes was inhibited in the absence of Myosin-IIA. This resulted from inefficient trans-endothelial migration (TEM) of Myosin-IIA depleted T cells; specifically, Myosin-IIA contractility was important to squeeze the body of the T cell (containing the nucleus) through the endothelial cell layer. Overall, our results suggest a critical role for Myosin-IIA in promoting TEM and migration through constrictive environments, while migration within more 'permissive' environments is not strictly dependent on the function of this myosin motor.

This work was supported by the NIH (RO1AI052116) and the Leukemia and Lymphoma Society.
Expression of chemokine receptors implicated in skin-homing on Herpesvirus-specific CD8+ T-cells (95.14)
Authors: Hensel Michael T , Caka Selin E , Wald Anna , Koelle David M
Published: January 2023

HSV-2-specific CD8+ T-cells express high levels of cutaneous lymphocyte associated antigen (CLA) and the related E-selectin ligand (ESL). These mediate rolling adhesion to inflamed dermal endothelium. EBV- or CMV-specific CD8+ T-cells express low levels of CLA/ESL. Full arrest and diapedesis are mediated via the chemokine axis. Chemokine receptors implicated in skin homing include CCR4, CCR6, CCR8, CCR10, and CXCR3. We examined cell surface expression of these receptors on pathogen-specific, tetramer-positive CD8 T-cells and bulk CD4 and CD8 T-cells, using both cryopreserved and fresh PBMC. Our results confirm that amongst CD4+ cells, CLA+ cells selectively express CCR4 and CCR10, and extend these findings to bulk CD8+ cells. We did not, however, observe selective enrichment of CCR4 or CCR10 on HSV-2-specific CD8+ T-cells compared to bulk CD4+ or CD8+ T-cells. In contrast, we measured higher CXCR3 expression on HSV-2-specific CD8+ T-cells when compared to bulk CD4+ and CD8+ T-cells, or EBV- or CMV-specific CD8+ T-cells. This suggests HSV-2-specific CD8+ T-cells may utilize CXCR3 expression to home to infected skin lesions.

Support from: NIH Grant AI30731 and STD/AIDS Research Training Grant 5T32AI007140
Interleukin-7 receptor (IL7R) expression provides the potential for long-term survival of both CD62Lhigh central memory T cells and Th1 effector cells during Leishmania major infection (96.6)
Authors: Colpitts Sara L , Dalton Nicole M , Scott Phillip
Published: January 2023

Infection with the protozoan parasite Leishmania major induces a state of concomitant immunity wherein secondary immunity is dependent upon the persistence of the original pathogen. Our lab has described two populations of L. major-induced CD4+ T cells that contribute to immunity: CD62Lhigh central memory T (TCM) cells and CD62Llow effector T (TEFF) cells. To determine if the prosurvival cytokine interleukin-7 (IL-7) contributes to maintaining these T cells, we examined IL-7 receptor (IL7R) expression on CD4+ T cells activated during L. major infection. We found that TCM cells present in chronically infected mice expressed high levels of the IL7R. However, we also found that CD62Llow cells responding to L. major infection expressed the IL7R. A large percentage of the IL7Rhigh CD62Llow cells were Th1 cells, based on transcription at the IFN-γ locus and upregulation of the Th1 transcription factor T-bet. The upregulation of T-bet did not prevent IL7R expression by L. major-responding CD4+ T cells nor did the absence of T-bet result in increased IL7R expression. Finally, blockade of IL7R signaling inhibited delayed type hypersensitivity responses in immune mice challenged with L. major, indicating that IL7R signaling contributes to the maintenance of Teff cells. Thus, both TCM and Th1 effector cells can express the IL7R during chronic L. major infection, which provides a potential means for their long-term survival.
IL-15 and IL-21 Mediated Homeostatic Proliferation of Human CD8 Memory T cells (96.7)
Authors: Nguyen Huy , Weng Nan-ping
Published: January 2023

Accumulation of CD28- CD8 T cells is a hallmark of the age-related decline in immune response. IL-15, a common γ-chain (γc) cytokine, is critical for the homeostatic proliferation of memory CD8 T cells and is recently shown to be associated with the down-regulation of CD28 expression. IL-21 is a newly identified member of the γc family of cytokines that has been demonstrated to have pleiotropic effects, influencing T cell proliferation and function. In IL-15-stimulated human CD8 naive T cells, IL-21 has been shown to prevent down-modulation of CD28 expression and augment cell growth. Here, we analyzed the effects of IL-21 on IL-15-stimulated memory CD8 T cells. In comparison between CD28+ and CD28- memory CD8 T cells under IL-15 alone or with IL-21 in vitro in young and old donors, we found that IL-21 drives more IL-15-stimulated cells to enter cell division and undergo apoptosis and that IL-21 preferentially enhances IL-15-induced proliferation of the CD28+ subset of memory CD8 T cells over its CD28- counterpart. Furthermore, we showed that IL-21 delays the IL-15-induced CD28 down-regulation at the cell surface and mRNA level for memory CD8 T cells. These findings demonstrate that IL-21 can increase proliferation of CD28+ cells and delay the generation of CD28- cells in IL-15-stimulated memory CD8 T cells, suggesting a possible role of IL-21 as an antagonist to the IL-15-induced increase in CD28- CD8 T cells that occurs during aging.
Functional Characterization of CBAP in Controlling Bim-independent Hematopoietic Homeostasis (97.14)
Authors: Chiang Yun-Jung , Yen Jong-Young Jeffrey
Published: January 2023

The lifespan of hematopoietic cells is involved in controlling inflammation and autoimmune response. CBAP (Common Beta chain Associating Protein) is a newly annotated membrane bound protein and is identified to associate with Box II domain, highly conserved among cytokine receptor superfamily, of common beta chain (βc) of GM-CSF/IL-3/IL-5 receptor.

In GM-CSF withdrawal-induced cell death assay, CBAP is demonstrated to trigger apoptotic signal, which is involved in Bax/Bak activation, and could be blocked by overexpression of Bcl-2 protein. In order to characterize the biological role of CBAP in vivo, we created the CBAP-deficient mice in either wild-type (wt) or Bim-deficient genetic background. We find that CBAP deficient mice are viable and fertile. The targeted allele could be transmitted according Mendel's Law inside the family, suggesting there is no apparent reproductive defect. In addition, we find the alterations of haematopoietic homeostasis. CBAP-deficiency increased the numbers of T cells, B cells and dendritic cells in the peripheral lymphatic organs in both the wt- and Bim-/- background, suggesting CBAP plays a non-redundant role in regulating hematopoietic homeostasis. CBAP-deficient primary bone marrow-derived dendritic cells (BMDCs) are more resistant to GM-CSF withdrawal-induced cell death, suggesting the contribution of CBAP in the regulation of apoptosis. Taken together, these results suggest the potential role of CBAP in controlling death of hematopoietic cells as well as playing a redundant role of Bim-regulated cell death.
Effects of IL-2 and IL-15 on CXCR6 Expression and Cytokine Production by Human Lymphocytes (98.26)
Authors: Perna Avi , Yang Xifeng , Li Linda
Published: January 2023

The chemokine receptor CXCR6, also known as BONZO and STRL133, is a seven transmembrane G protein-coupled receptor, primarily expressed on a subset of T effector cells, NK and NKT cells. Initially characterized as a co-receptor for HIV and SIV, studies of the interaction with CXCL16 have elucidated the important role of CXCR6 in leukocyte chemotaxis. This study focused on the kinetic effects of IL-2 and IL-15 on CXCR6 expression and cytokine production by lymphocytes in vitro. Freshly isolated human peripheral blood mononuclear cells (PBMCs) were cultured in the presence of recombinant IL-2 (20ng/mL) or IL-15(100ng/mL) for 15 days. The cells were harvested at regular time points and CXCR6 expression was analyzed via polychromatic flow cytometry. The results indicated that both IL-2 and IL-15 were able to effectively upregulate CXCR6 expression. Compared to non-stimulated cells, the frequencies of CXCR6+ cells were significantly increased (from 5-6% to 14-17%) at day 4 with maximum levels (30-50% CXCR6+) observed from day 7-9. The CXCR6 expressing cells were mainly in the lymphoblastic cell population and all CXCR6+ cells expressed CD3. We then further studied cytokine production profiles of the CXCR6 positive cells. After additional 6 hours re-stimulation with PMA plus ionomycin (in the presence of monensin), cytokine production was examined by intracellular staining and flow cytometric analysis. We found that IFN-g production by CXCR6+ cells (30-80%) was potentiated compared to their CXCR6- counterparts (10-30%). Additionally, IL-2 and IL-15 stimulation attenuated the ability of cultured PBMCs to produce IL-2. Further cytokine profiles are under investigation.
CXCL11 and CXCL12 activates ERK1/2 and NF-kB in breast cancer cells via CXCR3, CXCR4 and CXCR7 (98.3)
Authors: Mosley Lasharon  D , Singh Rajesh , Sharma Praveen K , Singh Shailesh , Lillard, Jr. James W
Published: January 2023

Metastasis of breast cancer (BrCa) cells involves organ-specific homing for migration, invasion, adhesion, and survival at secondary sites. Chemokines, such as stromal cell-derived factor-1 (SDF-1; CXCL12) and interferon-inducible T cell α-chemoattractant (I-TAC; CXCL11), and their receptors CXCR4, CXCR7 (CXCL11 & CXCL12), and CXCR3, respectively, are believed to play crucial roles in this process by G protein coupled signaling events leading to the activation of transcription factors. Our study investigates the CXCL12:CXCR4/CXCR7 as well as CXCL11:CXCR3/CXCR7 interactions on pathways known to mediate transcriptional activation of BrCa cell invasion mechanisms. ImageStream analysis revealed that treatment of MCF-7 and MDA-MB-231 cell lines with either CXCL11 or CXCL12 resulted in the activation and translocation of ERK1/2 and NF-¿B to the nucleus. FACE assay analysis demonstrated that stimulation with these chemokines selectively induced ERK1/2 and NFκB phosphorylation through FAK-, Src-, and/or PI3K-dependent pathways. These findings suggest CXCL11 and CXCL12 differentially induce signal transduction through CXCR3, CXCR4, and CXCR7. [This study was supported by funds from the James Brown Graham Cancer Center, Smith & Lucille Gibson Endowment. and National Institute of Health Grants CA092078, CA086359, and MD00525.]
Relationship between the high expression of nuclear beta-catenin in dendritic cells from NOD mice and the pro-inflammatory phenotype of these cells (99.18)
Authors: Atay Safinur , Villard Marine , Martin Michael , Parnell Sarah , Kosiewicz Michele , Alard Pascale
Published: January 2023

Pro-inflammatory cytokines are increased during the active stages of Type 1 diabetes and play an important role in disease development in NOD mice. Furthermore, antigen presenting cells from NOD mice produce elevated levels of IL-12 and IL-6 due to a hyperactive NFκB. β-catenin, is an important protein involved in gene regulation, and was recently found to interact physically with NFκB and regulate its translocation in the nucleus. We, therefore, have begun to investigate the relationship between β-catenin expression and the pro-inflammatory phenotype of dendritic cells (DC) in diabetes-susceptible NOD mice.

We have found a dramatic increase in the levels of nucleocytoplasmic β-catenin in bone marrow-derived DC, not macrophages, from NOD mice. Moreover, the increased levels of nuclear β-catenin correlated with increased levels of nuclear NFκB upon LPS stimulation. Altogether, these data indicate that NOD DC overexpress β-catenin, which upon accumulation in the nucleus may interact with NFκB and contribute to the pro-inflammatory phenotype of these cells. Experiments using β-catenin siRNA or inhibitors are ongoing to determine the role that β-catenin plays in the pro-inflammatory phenotype of NOD DC, and assess whether β-catenin may be a potential therapeutic target for the prevention and/or treatment of Type 1 diabetes. Funded by KDRB.
Analysis of regulatory and effector T cell repertoires during MOG-EAE (99.7)
Authors: Liu Xin , Steeves Meredith A , Nguyen Phuong , Geiger Terrence L
Published: January 2023

How the repertoires of conventional (Tconv) and Foxp3+ regulatory T-cells (Treg) compare in the context of active autoimmune disease is poorly understood. We assessed regulatory and effector T-cell repertoires in experimental allergic encephalomyelitis (EAE) induced by immunization with myelin oligodendroglial glycoprotein (MOG). We produced retroviral transgenic mice in which all T-cells express a single TCRá, and Treg are distinguishable from Tconv by a GFP-Foxp3 transgene. Repertoire was analyzed by cloning and sequencing the TCRâ chains from sorted CNS or splenic T-cells in mice with or without EAE. This demonstrated both substantial receptor diversity as well as public and private dominant receptors. Under inflammatory conditions, the Tconv used a repertoire distinct from that of Treg, though the repertoires overlapped. The extent of this overlap was not significantly increased with immunization or when comparing CNS infiltrating versus splenic T-cells. Further, greater similarity existed when comparing Treg or Tconv between spleen and CNS than when comparing the different cell types within an organ. This suggests that site-specific conversion between Treg and Tconv is limited in the context of active EAE. A subset of Vâ8.2, Jâ2.7 TCR were cloned, transduced into T-cell hybridomas, and functionally analyzed. Virtually all CNS-infiltrating Vâ8.2, Jâ2.7+ Tconv were MOG-specific whereas only ~half of the CNS Treg were. In the spleen, few TCR were MOG-specific. Therefore both MOG-specific Treg and Tconv with distinct repertoires are concentrated within the CNS during EAE, though infiltrating Treg seemingly possess a broader spectrum of antigen specificities.
Contribution of memory and naïve B cells to myelin specific T cell function in relapsing remitting multiple sclerosis (99.8)
Authors: Harp Christopher T , Frohman Elliot , Monson Nancy L
Published: January 2023

B cell depletion therapy reduces numbers of B and T cells within the cerebrospinal fluid (CSF) of relapsing remitting multiple sclerosis (RRMS) patients, suggesting that reduction of CSF B cells modulates the T cell pool in RRMS. We hypothesized that T-B cell interactions are required in order for myelin reactive T cells to proliferate and secrete cytokines optimally, and that memory B cells would be more effective APCs than naïve B cells, because of their higher expression of co-stimulatory molecules and high affinity B cell receptors. To test this hypothesis, we sorted naïve (CD19+CD27-) and memory (CD19+CD27+) B cells from the peripheral blood of 12 treatment naïve RRMS patients, incubated them with autologous purified T cells in the presence of myelin-derived proteins or control recall antigens, and measured T cell proliferation and cytokine production. Both naïve and memory B cells elicited CD4+ and CD8+ T cell proliferation and IFN-gamma secretion to recall antigens, although memory B cells from these patients elicited greater myelin specific CD4+ T cell proliferation than naïve B cells. These data suggest that in a subset of RRMS patients, the memory B cell pool has an enhanced ability to induce activation of myelin specific T cells, most likely because the memory B cell pool is enriched for B cells that have undergone myelin specific affinity driven maturation. This work was supported by Teva Neuroscience.
Immunological characterization of oncogene-driven models of hepatocellular carcinoma. (100.19)
Authors: Scarzello Anthony , Stauffer Jim , Back Tim , Subleski Jeff , Weiss Jonathan , Ortaldo John , Wiltrout Robert
Published: January 2023

Constitutively active AKT, MET, and beta-catenin (CAT) are initiating oncogenes that efficiently induce hepatic primary tumors when co-delivered; but not as single agents. Using a qPCR assay, we have begun to detail the early events of oncogene integration and to correlate the amount of integrated oncogene with developing tumor size. Furthermore, we found that the serum from mice injected with MET/CAT have elevated levels of alpha fetoprotein, a biomarker which has been similarly observed in advanced cases of HCC. Correlating the influx of infiltrating leukocytes and serum biomarker levels with the quantity of integrated oncogenes is critical to characterize their relative contributions to tumor progression. The profile of liver leukocytes in both MET/CAT and AKT/CAT murine models display phenotypes similar to those in HCC patients. By day 14, increased frequency and numbers of Tregs, MDSCs, as well as an up-regulation of PD-1 on CD4+ and CD8+ T cells was observed. The oncogene-driven tumor models described in this study exhibit many of the molecular and cellular events underlying HCC progression in humans. This molecularly defined approach to hepatocellular oncogenesis should enable us to characterize the contributions of inflammation in a primary tumor model with a defined oncogene signaling pathway.
Crystal structure of a gp100:44-59 peptide-HLA-DR4 complex enables design of an altered peptide ligand with enhanced immunogenicity (100.25)
Authors: Chen Shuming , Depontieu Florence , Li Yili , Salay Theresa , Mariuzza Roy , Topalian Suzanne
Published: January 2023

T cells recognizing tumor-associated self-antigens play a central role in anti-cancer immunity. CD4+ T cells are important in generating and regulating anti-tumor immunity, and MHC II-restricted tumor epitopes may enhance the clinical efficacy of polyvalent cancer vaccines. Gp100 is a non-mutated lineage-specific melanoma antigen containing the dominant HLA-DRB1*0401-restricted epitope gp100:44-59 with intermediate MHC binding affinity. The crystal structure of gp100:44-59 bound to HLA-DR4 was determined to 3 Å resolution to facilitate rational design of agonistic altered peptide ligands (APLs) which could improve the immunogenicity of this self antigen. The structure identified Leu48 as the peptide’s P1 MHC anchor, revealing suboptimal secondary anchors at P4, P7, and P9 based on published motifs. APLs with substituted secondary anchors were assessed for recognition by a gp100:44-59-specific CD4+ T cell clone, identifying P9 Q56A as an optimal APL with 10-fold enhanced T cell recognition. Fresh PBL from a DR4+ melanoma patient were twice stimulated in vitro with wild type (WT) or Q56A peptides and then assessed for peptide recognition (IFN-g ELISPOT). While WT-raised T cell cultures were non-specific, Q56A-raised cultures were specific for both Q56A and WT peptides. Q56A T cells also recognized DR4+, gp100+ melanoma cells (GM-CSF ELISA). These findings suggest that gp100:44-59 Q56A has improved immunogenicity and may warrant clinical testing as a melanoma vaccine.
Impaired tumoricidal activity in macrophages infiltrating intraocular tumors is caused by selective inhibition of inducible nitric oxide synthase (NOS2) (100.5)
Authors: Vicetti Miguel Rodolfo , Cherpes Thomas , McKenna Kyle
Published: January 2023

Transfer of ovalbumin (OVA) specific cytotoxic CD8+ T lymphocytes (OT-I CTL) into C57BL/6 mice produces rejection of OVA-expressing intradermal tumors in a NOS2-dependent manner, while intraocular OVA-expressing tumors are fully refractory to transfer therapy. We have previously found that infiltrating CTL were not suppressed in the ocular tumor microenvironment. However, macrophages (Mϕ) from CTL-transferred ocular tumors were not tumoricidal in vitro due to reduced NO production, despite high levels of NOS2 protein. We could replicate this phenomenon in vitro, when peritoneal and RAW264.7 Mϕ were stimulated with IFN-γ and TNF or LPS in the presence of aqueous humor (AqH). Moreover, we found decreased NOS2 enzymatic activity in lysates of these Mϕ, which was not due to increased Arg1 activity or inhibited GTP cylohydrolase activity since AqH did not affect their mRNA levels, and supplementation with L-arginine and tetrahydrobiopterin did not overcome AqH-mediated NO suppression. Interestingly, AqH did not affect the expression of NOS2, Arg1, CD40, PDL-1, MHC-II, IL-12 and TNF, indicating selective inhibition of NOS2 activity. Finally, TGF-β2, CGRP and α-MSH alone or in combination did not recapitulate the suppressive effect of AqH on NO production by Mϕ. In conclusion, reduced NO production by ocular tumor associated Mϕ was due to inhibition of NOS2 activity through a post-translational mechanism, suggesting that the ocular microenvironment can modulate Mϕ effector function.
A phosphorylated β-catenin peptide that is presented by HLA-A2 generates strong phosphospecific T cell responses against melanoma (101.10)
Authors: Obeng Rebecca , Le Poole I. , Hunt Donald , Engelhard Victor
Published: January 2023

Using metal ion affinity chromatography and mass spectrometry, we have previously shown that phosphorylated peptides are endogenously processed and presented by many different MHC I and II molecules, and have identified differentially displayed phosphopeptides presented by HLA-A2 on cancer cells. Many of the source proteins for these phosphopeptides are involved in tumorigenesis and metastasis. These peptides are appealing immunotherapeutic targets since mutations in or downregulation of the source proteins as a means of immune escape may compromise malignancy. We focused on one phosphopeptide spanning residues 30-39 of β-catenin (βcatpS33). When used to immunize HLA-A2 transgenic mice, βcatpS33 was very weakly immunogenic. Replacement of a suboptimal Ala anchor residue at P10 with a Val (βcatpS33(V)) enhanced the HLA-A2 binding affinity 10 fold and substantially improved immunogenicity. CD8 T cells from βcatpS33(V)-immunized mice were crossreactive to βcatpS33. The T cells also recognized endogenously processed βcatpS33 on several HLA-A2+ melanoma cells. Thus, crossreactivity to the natural βcatpS33 phosphopeptide can be maintained by making minimal changes in the sequence while preserving the ability of T cells to discriminate between the phosphorylated and unphosphorylated forms of the peptide. Enhancing immunogenicity by fine-tuning such an attractive tumor antigen linked to tumorigenic and metastatic processes should improve the efficiency of tumor control.
Mutations in BRCA-1 result in the functional loss of NKT cells (101.18)
Authors: Webb Tonya , Hester Danubia , Jones Laundette
Published: January 2023

Natural killer T (NKT) cells have been shown to be important in regulating immune responses to tumors; however, the number and function of NKT cells are reduced in cancer patients. Genetic factors regulating NKT cell number and function have yet to be determined. The Breast Cancer Susceptibility gene 1 (BRCA-1) encodes a tumor suppressor gene that functions, in part, as a caretaker gene in preserving chromosomal stability. In this study we have investigated the role of BRCA-1 on NKT expression and function using a transgenic mouse model in which BRCA-1 is mutated. This mouse model has a direct translational link to a specific type of human breast cancer, because it develops the same genetic changes that are described in BRCA-1-related human cancer. BRCA-1 is highly expressed in the T cell lineages, and we have found that our NKT cell lines express BRCA-1 protein. When we examined the liver and spleens of BRCA-1 mutant mice, we found that the NKT cell population was significantly reduced, compared to wildtype control mice. The classical CD4 and CD8 T cell subpopulations were comparable between the BRCA-1 mutant mice and wildtype controls, which suggests that NKT cell population is specifically regulated BRCA-1. Collectively, our data suggest that determining whether genetic susceptibility alters the function of NKT cells, a key regulator of anti-tumor immune responses, could help in designing novel therapeutics targeting aggressive breast cancers.
Toll-like receptor agonists to the rescue: saving tumor-specific T-cells (101.8)
Authors: Srivastava Ratika , Zheng Liquin , Geng Degui , Davila Eduardo
Published: January 2023

The molecular basis for the costimulatory effects of toll-like receptor (TLR) agonists are being gradually unravelled, adding to our understanding of how TLR signals enhance T-cell-mediated immune responses. Recent studies indicate that the adjuvant effects of certain TLR agonists may be attributed to activating TLR-myeloid differentiation factor (MyD88) directly in T-cells. We examined the survival of TLR2-MyD88-stimulated CD8 T-cells derived from melanoma patients and T-cell receptor transgenic pmel mice. TLR2 stimulation on pmel CD8 T-cells but not on TLR2-/-pmel or MyD88-/-pmel T-cells enhanced T-cell expansion primarily by reducing apoptosis rather than enhancing cell division. We determined the requirement for intrinsic MyD88 signals within pmel T-cells, by co-transferring congenic MyD88-/-pmel and pmel T-cells into CD45.1 mice and comparing T-cell numbers at different time points. Pmel T-cells outnumbered MyD88-/-pmel T-cells as early as 14 days after adoptive cell transfer and became more disparate after 34 days. MyD88-/- T-cells could not be rescued despite conditions sufficient to support pmel T-cell survival within the same environment. At the molecular level, we detected significant changes in various apoptotic molecules, favoring T-cell longevity. These findings reveal a novel role for TLR-MyD88 signals within tumor-specific T-cells which may inspire new approaches for enhancing cancer immunotherapies by manipulating TLR-MyD88 signaling within T-cells.