bluuebunny/crossref_metadata_2025 huggingface dataset:
T cell epitopes derived from autoantigens are selected differently than those from exogenous antigens (130.15)
Authors: Kim Ae , Hartman Isamu , Boronina Tatiana , Cole Robert , Sadegh-Nasseri Scheherazade
Published: January 2023

We have established a novel minimalist cell-free MHC class II antigen-processing system for identifying immunodominant epitopes from protein antigens. The system constitutes HLA-DR1, HLA-DM (DM) and three cathepsins. This system not only identifies the immunogenic epitopes from complex protein antigens, but also provides insight to the mechanism of immunodominance. Understanding of how these immunogenic epitopes are selected certainly provides better strategies for developing effective peptide vaccine designs and therapies against infectious diseases, cancer, autoimmune diseases and allergy. Using our cell-free system, and based on the source of the antigens categorized as exogenous antigens versus autoantigens, we find that both DM and cathepsins are crucial in regulating the selection of immunodominant epitopes. Three types of determinants are generated: first, non-dominant epitopes that are sensitive to both DM mediated dissociation, and cathepsins digestion. These epitopes were eliminated immediately from peptide repertoire. Second, T cell epitopes derived from the exogenous antigen are DM resistant and cathepsin sensitive. Third, T cells epitopes derived from an antoantigen that are sensitive to DM mediated dissociation, but resistant to cathepsin digestion. This shows that generation of immunodominant epitopes is regulated differently depending on the source of proteins antigens.
CD23-bound IgE augments and dominates recall responses through human naïve B cells (130.3)
Authors: Griffith Qyana , Liang YanMei , Onguru Daniel , Shah Taslima , Haas Helmut , Mwinzi Pauline , Ganley-Leal Lisa
Published: January 2023

Peripheral blood human naïve B cells express high levels of CD23 and circulate pre-loaded with IgE. The antigen specificity of CD23-bound IgE presumably differs from the B cell receptor (BCR) and may reflect the antigen specific mix of free serum IgE. CD23-bound IgE is thought to enhance B cell antigen presentation raising the question of how a B cell might respond when presented with a variety of antigens and CD23-bound IgE specificities. We recently reported that an increase in CD23+ B cells is associated with resistance to schistosomiasis highlighting the potential importance of CD23-bound IgE in immunity. We sought to determine the relationship between BCR and CD23-bound IgE mediated B cell activation in schistosomiasis. Crude schistosome antigens downregulated basal B cell activation levels in individuals hyper-exposed to infectious worms. However, schistosome-specific IgE from resistant, occupationally exposed Kenyans recovered responses of naïve B cells to schistosome antigen. Furthermore, cross-linking of CD23 overrode intracellular signals mediated via the BCR illustrating its dominating role in B cell activation. Notably, the nature of the cognate antigen appeared to dictate the threshold of antigen-specific CD23-bound IgE required for B cell activation suggesting that CD23-bound IgE functions as a rheostat for B cell responses to antigenic stimuli. These results suggest that CD23-bound IgE augments and dominates host recall responses through naïve B cells.
Cutaneous delivery of Poly(dG) conjugated protein antigens targets and activates DCs in vivo. (130.38)
Authors: Toussaint Helene , Erdos Geza , Williams Melissa , Donahue Cara , Falo Louis
Published: January 2023

Efficient and specific targeting of exogenous antigen to antigen presenting cells (APCs) in vivo is a critical challenge for vaccine design. APCs acquire antigens from a variety of sources and endocytic mechanisms can predetermine intracellular antigen routing, processing and presentation to CD4+ or CD8+ T cells. We have designed an APC targeting strategy based on simple and direct coupling of Poly(dG) oligo deoxynucleotides to protein antigens. In comparison to soluble OVA, we find that conjugates of Poly(dG) and OVA (OVA-Poly(dG)) are rapidly internalized by dendritic cells (DCs) and follow distinct intracellular processing pathways. Functionally, OVA-Poly(dG) is both presented to OVA-specific CD4+ T cells (OT-II cells) and efficiently cross-presented to OVA-specific CD8+ T cells (OT-I cells) by DCs in vitro. Importantly, immunization of naïve animals by direct injection of OVA-Poly(dG) results in efficient priming of potent antigen specific CTL responses. Extending our findings to human skin, we show that I.D. injection of antigen-Poly(dG) conjugates results in efficient uptake by human skin migratory DCs including Langerin+ DCs that also acquire potent T cell allostimulatory function. Our data suggest that Poly(dG) can both target antigens to human skin immune APCs and function as an adjuvant to induce potent CD4 and CD8 mediated T cell immunity.
Naïve tumor-specific CD4+ T cells differentiated in vivo eradicate established melanoma (131.12)
Authors: Antony (PI) Paul , Xie Ying , Akpinarli Akgul , Maris Charles , Hipkiss Edward , Lane Malcolm , Kwon Eun-Kyung , Muranski Pawel , Restifo Nicholas
Published: January 2023

In vitro differentiated CD8+ T cells have been the primary focus of immunotherapy of cancer with little focus on CD4+ T cells. Immunotherapy involving in vitro differentiated T cells given after lymphodepleting regimens significantly augments antitumor immunity in animals and human patients with cancer. However, the mechanisms by which lymphopenia augments adoptive cell therapy and the means of properly differentiating T cells in vitro are still emerging. We demonstrate that naïve tumor/self-specific CD4+ T cells naturally differentiated into T helper type 1 (Th1) cytotoxic T cells in vivo and caused the regression of established tumors and depigmentation in lymphopenic hosts. Therapy was independent of vaccination, exogenous cytokine support, CD8+-, B-, NK and NK-T cells. Proper activation of CD4+ T cells in vivo was important for tumor clearance as naïve tumor-specific CD4+ T cells could not completely treat tumor in lymphopenic common gamma chain deficient (γc-/-) hosts. γc signaling in the tumor-bearing host was important for survival and proper differentiation of adoptively transferred tumor-specific CD4+ T cells. Thus, these data provide a platform for designing immunotherapies that incorporate tumor/self-reactive CD4+ T cells.
In vivo administration of artificial Antigen Presenting Cells licenses low avidity T cells for treatment of cancer (131.13)
Authors: Oelke Mathias , Ugel Stefano , Zoso Alessia , De Santo Carmela , Li Yu , Marigo Ilaria , Zanovello Paola , Scarselli Elisa , Cipriani Barbara , Bronte Vincenzo , Schneck Jonathan P.
Published: January 2023

The development of effective anti-tumor immune responses is normally constrained by low avidity, tumor-specific cytotoxic T lymphocytes (CTL) which are unable to eradicate the tumor. Therefore, new strategies to rescue anti-tumor activity of low avidity melanoma-specific CTL in vivo may improve immunotherapy efficacy. To boost the in vivo effectiveness of low avidity CTL we immunized mice bearing metastatic lung melanoma with artificial Antigen Presenting Cells (aAPC), made by covalently coupling pepMHC-Ig dimers and B7.1-Ig molecules to magnetic beads. aAPC treatment induced significant tumor reduction in a mouse telomerase antigen system and complete tumor eradication in a mouse TRP-2 antigen system, when low avidity CTL specific for these antigens were adoptively transferred. In addition, in an in vivo treatment model of subcutaneous melanoma, aAPC injection also augmented the activity of adoptively transferred CTL and significantly delayed tumor growth. In vivo tumor clearance due to aAPC administration correlated with in situ proliferation of the transferred CTL. In vitro studies showed that aAPC effectively stimulated cytokine release and TCR down-regulation in low avidity CTL. Therefore, in vivo aAPC administration represents a potentially novel approach to improve adoptive immunotherapy of cancer.
Development of an inducible MyD88.CD40 gene switch for potent anti-tumor dendritic cell (DC) vaccines (131.22)
Authors: Narayanan Priyadharshini , Lapteva Natalia , Seethammagari Mamatha , Slawin Kevin , Spencer David
Published: January 2023

We previously generated an inducible CD40 (iCD40) costimulatory switch enabling prolonged CD40 signaling in DCs in a ligand-independent manner. However, follow-up studies showed the requirement for an additional danger stimulus, lipopolysaccharide, for complete DC activation and licensing. To overcome drawbacks of systemic toxicity associated with endotoxin in a clinical trial, we proposed the development of a synthetic drug-inducible TLR4. Toward this goal, we have generated an inducible version of downstream adaptor MyD88 and combined it with iCD40 to generate a unified, inducible composite receptor, iMyD88.CD40. In vitro, in addition to upregulating maturation markers, iMyD88.CD40 activation by dimerizing drug led to elevated IL-12 production by both mouse and human DCs, prolonged DC survival and improved T cell responses to endogenous tumor antigens. Increased magnitudes of p38, IKK and JNK activation along with early activation of NF-kB family members, RelA, RelB and c-Rel form the molecular basis for the observed synergistic effects of iMyD88.CD40. In vivo, iMyD88.CD40-activated DCs migrated more efficiently to draining lymph nodes, promoted Th1 responses and abolished pre-established E.G7-OVA tumors and the less immunogenic B16 tumors. In conclusion, we have generated a novel chimeric DC activation switch combining signaling elements from adjuvant receptors enabling prolonged DC activation both ex vivo as well as in vivo following DC and drug delivery.
A persistent virus vector confers superior anti-tumor immunity,compared with a non-persistent vector (131.26)
Authors: Zhang Weijun , Zhang Tony , Turk Mary-Jo
Published: January 2023

Active vaccination strategies using viral vectors often give disappointing protection from tumor development, and usually require multiple immunizations. These approaches normally use viruses that cause acute infections, as they provoke potent CD8 T cell responses. Persistent virus vectors have not been used in this setting due to the perception that exhaustion of the T cell response occurs and would lead to poor anti-tumor protection. However, such exhaustion generally only occurs in high-load virus infections, whereas T cell function is intact in lower-load persistent infections. In fact, CD8 T cell responses in these infections, which are adapted for long-term immune surveillance,have properties that may make them more desirable for long-term anti-tumor immunity. In this report, we show that a persistent gammaherpesvirus vector provides superior protection against melanoma, relative to a nonpersistent mutant of the same virus. These data suggest that vaccine vectors derived from persistent viruses may perform better than those from acute viruses at mediating anti-tumor protection.
Targeting A2aR as a means of enhancing anti-tumor vaccines. (131.3)
Authors: Alme Angela , Senaldi Liana , Waickman Adam , Zarek Paul , Powell Jonathan
Published: January 2023

Adenosine, acting via the A2A adenosine receptor (A2aR), is emerging as an important inhibitor of immune function. Indeed, the tumor microenvironment contains relatively high levels of adenosine suggesting that tumor-derived adenosine is one mechanism by which cancers evade immune destruction. To better understand this potential mechanism of tumor immune evasion we challenged A2aR null mice with EL-4 lymphoma. In this model the A2aR null mice routinely rejected their tumors while Wildtype (Wt) mice succumb to tumor-induced death. Interestingly, while both Wt and A2aR null mice were able to reject low doses of tumor, when such mice were rechallenged with lethal doses of tumor, the A2aR mice were greatly protected compared to Wt controls. Such findings suggest that blocking the A2aR might greatly enhance the anti-tumor memory response. As such Wt and A2aR null mice were vaccinated with a whole cell GM-CSF secreting vaccine (GVAX) and challenged with B16 melanoma cells. In these experiments metastasis was markedly decreased in the untreated A2aR null mice when compared with untreated wt mice. Further, while the vaccine led to decreased metastasis in the Wt mice, the vaccine virtually eliminated lung metastasis in the A2aR null mice. Finally, pharmacologic blockade of the A2aR receptor in tumor-bearing mice promoted an increase in tumor antigen specific immunity. Overall, our data suggests that pharmacologic A2aR blockade can enhance the efficacy of tumor immunotherapy.
Activation of omental immune aggregates: a potential tumor therapy (131.37)
Authors: Sedlacek Abigail , Gerber Scott , Lord Edith
Published: January 2023

Tumor metastasis within the peritoneal cavity is a concern for those diagnosed with cancers of peritoneal organs. A tissue implicated in this process is the omentum, which is composed of adipose tissue embedded with immune cell aggregates. We, and others have demonstrated that upon introduction of tumor cells into the peritoneal cavity, omental immune aggregates are the initial site of attachment. Due to their high vascularity, they provide an ideal microenvironment for tumor growth. Thus, treatment of peritoneal metastases often includes removal of the omentum. However, we hypothesize that stimulation of omental immune cells might result in reduced tumor growth. Our initial experiments demonstrate that injection of particles into the peritoneal cavity resulted in their uptake by macrophages present in the peritoneal fluid and rapid migration to immune aggregates. To determine if tumor antigens would result in effective immunity when bound to these aggregates, mice were immunized intraperitoneally with irradiated EMT6 tumor cells and later challenged with live EMT6/GFP cells. Omenta and peritoneal fluid were analyzed for the presence of memory CD8+ T cells and tumor cells. Our results show that immunization results in increased memory CD8+ T cells and decreased tumor burden on the omentum, indicating that omental immune cells are capable of responding against tumor cells. Further studies are being done to examine tumor burden in mice immunized after tumor establishment.
Anti-melanoma immunity promoted by cutaneous genetic immunizations requires agonistic signaling via the nerokinin-1 receptor (131.6)
Authors: Mathers Alicia , Tkacheva Olga , Janelsins Brian , Schufeski William , Morelli Adrian , Larregina Adriana
Published: January 2023

In spite of initial enthusiasm provided by melanoma immunizations, the incidence of melanoma has increased in the past decade. To improve current melanoma vaccines is imperative that we understand the mechanisms involved in effective immunity to self-melanocyte Ags. The pro-inflammatory and immune functions of the skin are ideal for administration of cancer vaccines able to bias anti-tumor type-1 immunity. Pro-inflammatory neuropeptides that signal via the neurokinin-1 receptor (NK1R) expressed in skin cells, promote CD4+Th1 and CD8+Tc1 immunity to foreign Ag. Here, we analyzed the effects of signaling the NK1R during a skin genetic vaccination for melanoma. We utilized the gene gun to deliver plasmid DNA encoding murine TRP-2 with or without the NK1R agonist [Sar9Met O2)11]-SP (SarSP) to C57BL/6 mice. The need of the NK1R for anti-melanoma immunity was addressed by administration of our vaccine to NK1R-/- mice. GG immunizations in the presence of SarSP promoted TRP-2 immunity including short term-effector-, central- and effector- memory Th1 and CTLs resulting in the prevention of melanoma growth and long term survival of mice. Administration of TRP-2 vaccines to NK1R-/- mice showed an impaired ability to initiate anti-TRP-2 immunity. Our results shed light on the mechanisms involved in induction of self-Ag specific immune responses capable of overcoming tumor growth, and provide the foundation for the development of an effective melanoma vaccine.
The role of reactive oxygen intermediates (ROI) in B cell activation, proliferation, and differentiation (132.10)
Authors: Crump Katie , Grayson Jason
Published: January 2023

Reactive oxygen intermediates (ROI) are generated during mitochondrial respiration, receptor ligation, and microorganism digestion. Ligation of the BCR resulted in ROI production that reaches a maximum by 15 minutes. ROI can modify all macromolecules, but we are interested in reversible oxidation events that modulate signaling. Our focus is on cysteine sulfenic acid, the first cysteine oxidation product. Using 5,5 dimethyl-1,3-cyclohexanedione (dimedone), a compound that covalently reacts with sulfenic acid to prevent further oxidation or reduction, we addressed the role of reversible sulfenic acid formation in mature B cell activation, proliferation and differentiation. Following incubation with antibodies against surface bound Immunoglobulin M (IgM), mature B cells divide several times by three days. Incubation with dimedone results in a concentration dependent block of the initial division. This contrasted with dimedone-pretreated CpG DNA and LPS-stimulated B cells, where subsequent divisions were reduced compared to vehicle-treated cells. Dual stimulation with BAFF, CD40, and IL-4 did not rescue the effect of dimedone on proliferation. Cells pretreated with dimedone did not flux calcium when incubated with IgM, and in vitro plasma cell differentiation was also decreased. These studies demonstrate that mature B cell activation, proliferation and differentiation require reversible cysteine sulfenic acid formation.
The bone marrow environment maintains polyfunctional memory T cells during human aging (132.12)
Authors: Herndler-Brandstetter Dietmar , Landgraf Katja , Brunauer Regina , Jenewein Brigitte , Tzankov Alexandar , Keller Michael , Kloss Frank , Gassner Robert , Lepperdinger Günter , Grubeck-Loebenstein Beatrix
Published: January 2023

The bone marrow (BM) has recently been attributed a key role in memory T cell maintenance. However, little is still known about the distribution, phenotype and function of human T cell subsets in the BM and how aging affects BM T cell function and survival. Here we show that human BM T cells are in a heightened activation state and express a characteristic set of chemokine- and co-stimulatory receptors compared to peripheral blood (PB). BM T cells also rapidly proliferate upon stimulation with anti-CD3 and IL-15. Moreover, the number of polyfunctional CD4 and CD8 T cells was higher in the BM compared to the PB. Aging led to a decline of naive T cells and to an accumulation of cells with an effector/effector-memory phenotype in the BM. Though, the age-related increase in highly differentiated CD57-expressing effector CD8 T cells was much lower in the BM compared to the PB. During aging, the frequency of polyfunctional BM T cells was maintained. The expression of IL-6 was increased during aging in the BM and together with IL-15 might contribute to the sustained activation of memory T cells in the BM. Gene array analysis also revealed a potential cytokine network responsible for the age-related increase in IL-6 expression. In conclusion, our results indicate that the BM environment is important for the maintenance of polyfunctional memory CD4 and CD8 T cells during human aging.
The A2A Adenosine Receptor regulates T cell effector and memory differentiation (132.13)
Authors: Waickman Adam , Powell Jonathan
Published: January 2023

The A2A adenosine receptor (A2A AR) plays a critical role in regulating immune system activation and tolerance. We hypothesized that A2AR blockade would enhance T cell responses to viral infection. Indeed, A2A AR-/- T cells demonstrate increased expansion in response to vaccinia infection when measured 5 days after infection, and in co-adoptive transfer experiments A2A AR-/- clonotypic cells out perform their WT clonotypic counterparts. Surprisingly, two weeks post infection such was not case. Instead we found fewer A2A AR-/- clonotypic cells. Interestingly, in spite of the fact that the vaccinia infection is cleared rapidly, the A2A AR-/- clonotypic cells appeared “exhausted” displaying markers of chronic activation and the inhibitory receptor PD-1. Unlike what was seen with the A2A AR-/- T cells, pharmacologic treatment of WT mice with an A2A AR antagonist during the 5 days post infection led to a marked increase recall potency. Overall, our data demonstrate a previously unappreciated role for adenosine and the A2aR in regulating effector cell expansion and memory differentiation. Consequently, pharmacologic A2A AR blockade selectively during the perivaccine period can lead to both enhanced effector and memory responses.
Origin of Thymic Adipocytes in Aging: Incidental to Thymopoiesis or Instigator of Immunosenescence? (132.22)
Authors: Dixit Vishwa , Youm Yun-Hee , Yang Hyunwon , Venkov Christo , Manley Nancy , Nielson Eric , Leff Todd , Vandanmagsar Bolormaa
Published: January 2023

Vishwa Deep Dixit, Yun-Hee Youm, Hyunwon Yang, Christo Venkov, Nancy R Manley, Eric Nielson, Todd Leff, Bolormaa Vandanmagsar By 50 years of age most of human thymus is replaced with adipocytes of unknown provenance and uncertain function. We investigated the lineage and mechanism of thymic adipocyte formation during aging and asked whether manipulating these pathways regulates thymic function. Using.We demonstrate that indelibly LacZ marked FoxN1 thymic epithelial cells transition to give rise to local tissue fibroblasts via the epithelial-mesenchymal transition(EMT)process. Importantly, we found an age-related increase in pro-EMT regulator FSP1 and pro-adipogenic transcription factor PPARγ in thymus. Consistent with these findings, a subset of FoxN1LacZ+ cells are FSP1+ with large unilocular lipid droplet, typical of adipocyte phenotype. Furthermore, aged FSP1.GFP reporter mice revealed that the sorted FSP1+ thymic fibroblasts lack TEC signatures and express PPARγ, indicative of adipogenic lineage commitment. Notably, overexpression of constitutive-active PPARγ in FSP1+ adipogenic lineage cells led to increased thymic adipogenesis and reduced thymopoiesis. Conversely, inhibition of EMT and thymoadipogenesis via anti-aging intervention, caloric restriction prevented thymic aging and immunosenescence. Future longevity studies using specific genetic manipulation of these pathways may provide a definitive answer to the puzzle of age-related thymic adiposity and involution.
Gene Signature of Memory CD8+ T cells Produced by Irradiated Plasmodium Sporozoite Immunization (132.28)
Authors: Tse Sze-Wah , Chen Yun-Chi , Cockburn Ian , Zavala Fidel
Published: January 2023

In murine malaria models, liver resident memory CD8+ T cells are important for protective immunity as they efficiently eliminate malaria infected hepatocytes in vivo. Further characterization of liver resident memory CD8+ T cells is therefore very crucial for the design of an effective anti-malaria vaccine. Here, we describe a microarray approach aims to elucidate the genes and pathways that may involve in the homing, function and differentiation of liver resident memory T cells. We compared the gene expression profiles of antigen specific CD8+ T cells sorted from liver and those from spleen of mice 30-45 days after immunization with irradiated Plasmodium yoelii sporozoites. Preliminary analysis suggests memory CD8+ T cells from liver and spleen have very distinct gene expression profiles. We found clusters of genes enriched in biological functions such as adhesion, metabolic process, cell cycle and signal transduction. The results suggest that CD8+ T cells reside in peripheral organs such as the liver, may have undergone a different program of development than those memory cells that reside in secondary lymphoid organs such as the spleen.
Chronic alcohol consumption compromises NK cell release from the bone marrow and impairs NK cell maturation in the spleen (132.32)
Authors: Zhang Hui , Zhu Zhaohui , Meadows Gary
Published: January 2023

NK cells primarily originate in the bone marrow (BM). The release of these cells from BM to peripheral organs is associated with the sphingosine 1-phosphate (S1P) and S1P receptor 5 (S1PR5) signaling system. T-bet controls the expression of S1PR5 on NK cells. Chronic alcohol consumption inhibits splenic NK cell cytolytic activity and compromises NK cell release from the BM. The underlying mechanism is still unknown. Herein, we used a murine model to study the effects of chronic alcohol consumption on NK cell development in BM and spleen. Alcohol consumption increased NK cells in the BM. The increased cells were CD27hiMac-1hi. The expression of S1P5 and T-bet in splenic and BM NK cells did not change in the alcohol consuming mice. The number of NK cells in the spleen was significantly decreased. The decreased NK cells were CD27loMac-1hi. The percentage and numbers of the activating receptor, Ly49D and Ly49H, positive NK cells decreased significantly. However, the decrease in the percentage and number of the inhibitory receptor, Ly49G, Ly49C, and Ly49A, expressing NK cells was lower than the activating receptor positive NK cells. These results indicate that chronic alcohol consumption blocks NK cell release from BM at the CD27hiMac-1hi stage. The block is independent of T-bet and S1PR5 expression. The results also indicate that alcohol consumption inhibits terminal maturation of NK cells before they acquire activating Ly49 receptors in the spleen.
RANK-L expression marks long-lived plasma cells (132.5)
Authors: Quinn William , Stohl William , Choi Yongwon , Cancro Michael
Published: January 2023

Plasma cells (PC) are the effectors of humoral immunity. Short-lived PC are generated rapidly after either thymus-independent (TI) or thymus-dependent (TD) immunization, yielding low affinity antibodies associated with early primary immune responses. In contrast, the lasting protective titers of isotype switched, high affinity antibodies associated with TD responses are maintained by long-lived PC. These cells persist indefinitely in the bone marrow (BM), suggesting that unique homing properties, survival requisites, and stromal interactions may underlie their protracted survival. Herein we show that TD PC express Receptor Activator for Nuclear Factor κ B Ligand (RANK-L, also termed TRANCE), whereas TI generated PC do not. In addition, in vivo administration of RANK-Fc leads to a profound reduction in pre-existing long-lived plasma cell pools, and prevents the establishment of long-lived PC following primary immunization. Further, mixed bone marrow chimeras in which B lineage cells lack RANK-L display marked impairment in the generation of isotype switched splenic and BM PC following TD immunization. Finally, RANK-L expression enables TD PC to induce APRIL production by myeloid cells, which in turn fosters PC survival. Together, these findings reveal a novel circuit whereby RANK-L-expressing PC both induce and maintain their own survival niche, and establish RANK-L as a definitive marker for long-lived PC and their progenitors.
Role of G-protein Coupled Receptor Kinases GRK2 and GRK3 in C3a Receptor Signaling in Human Mast Cells (133.12)
Authors: Guo Qiang , Subramanian Hariharan , Ali Hydar
Published: January 2023

Studies with animal models have shown that complement components C3a and C5a play an important role in the effector phase of allergic asthma. We have previously shown that C3a activates human CD34+-derived mast cells to induce degranulation. To determine the role of GRKs on C3a receptor (C3aR) signaling in human mast cells, we used shRNA to knockdown GRK2 and GRK3 a human mast cell line, HMC-1 and CD34+-derived cultures of primary mast cells. We found that GRK2 and GKR3 knockdown inhibited C3aR desensitization as measured by enhanced C3a-induced Ca2+ mobilization in HMC-1 cells and greater degranulation in CD34+ mast cells but had no effect on receptor internalization. In addition, GRK3-knockdown in HMC-1 cells showed increased expression of cell surface C3aR and this was correlated with enhanced extracellular-signal-regulated kinase (ERK) activation and a four-fold increase in C3a-induced chemotaxis. Surprisingly, there was no enhancement of these responses in GRK2 knockdown mast cells. These findings suggest that while GRK2 and GRK3 are required for C3aR sensitization, presumably by promoting receptor phosphorylation, they play distinct roles in modulating downstream signaling such as ERK phosphorylation and chemotaxis.
Human natural killer cells express functional formyl peptide receptors (133.13)
Authors: Bae Yoe-Sik , Kim Sang Doo , Kim Jung Mo , Lee Ha Young , Shim Jae Woong , Kim Hak Jung , Seo Su-Kil , Yun Jeanho
Published: January 2023

We tested the expression of the formyl peptide receptor (FPR) family and the functional roles of the FPR family in human natural killer (NK) cells. All tested human NK cells express two members of the FPR family (FPR1 and FPR2). The expression of FPR3 was noted to occur in a donor-specific manner. The stimulation of NK cells with FPR family-selective agonists (fMLF, MMK-1, F2L, and WKYMVm) elicited cytolytic activity in resting NK cells, but not in IL-2 activated NK cells; the cytolytic activity was not inhibited by pertussis toxin (PTX). The FPR family agonists also stimulated chemotactic migration of IL-2-activated NK cells, but not resting NK cells; the chemotactic migration was completely inhibited by PTX. WKYMVm stimulates ERK, p38 MAPK, and JNK activities in both resting and IL-2-activated NK cells. WKYMVm-induced chemotactic migration was partially inhibited by PD98059; however, the inhibition of JNK by its selective inhibitor (SP600125) dramatically inhibited the WKYMVm-induced cytolytic activity. Our findings indicate that human NK cells express functional members of the FPR family, and in turn the activation of the three members of the FPR receptor family elicit cytolytic activity in NK cells. We suggest that FPR family are potentially important therapeutic targets for the modulation of NK cell-mediated immune responses.
Role of CXCL12 and its receptor, CXCR4, in diversification of the rabbit primary antibody repertoire (133.3)
Authors: Lanning Dennis , Knight Katherine
Published: January 2023

The antibody repertoire of early postnatal rabbits is of limited diversity due to preferential V gene usage during VDJ gene rearrangement. Early in life, rabbit B cells expand this repertoire by diversifying their VDJ genes in gut-associated lymphoid tissue. VDJ gene diversification begins around 1 week of age, in B cells located in the basolateral region of nascent follicles. Because these B cells share similarities with B cells in germinal center dark zones, we hypothesized that activated B cells localize to the basolateral region in response to the chemokine CXCL12. To test this hypothesis, we examined B cell expression of CXCR4, the CXCL12 receptor, by in situ hybridization. We found that B cells in the basolateral region, but not those located elsewhere in the follicle, expressed CXCR4. Because antibody repertoire diversification requires signals derived from intestinal commensals, we further examined CXCR4 expression in appendices that were kept sterile by surgical ligation at birth. We found that B cells in these appendices did not express CXCR4 and did not diversify their VDJ genes. By contrast, we detected CXCL12 expression in both conventional and sterile appendices, demonstrating that it is not dependent on commensal-derived signals. Our results suggest that B cells activated by microbial-derived signals upregulate CXCR4 and migrate to the basolateral region of the follicle, likely in response to CXCL12, where they proliferate and diversify their VDJ genes.
CXCR6 mediates migration and invasion of breast cancer cells in a Src, FAK, and ERK1/2 dependent fashion (133.6)
Authors: Triplett Ashley , Sharma Praveen , Singh Rajesh , Lillard James , Singh Shailesh
Published: January 2023

Standard treatment modalities for breast cancer has improved the outlook for women afflicted with it. However, 40% of breast cancer patients still die from the disease, primarily due to metastasis. In this regard, chemokines and their corresponding receptor interactions in metastasis is an area of intense pre-clinical and clinical research. Our lab has demonstrated higher expression of CXCR6 in breast cancer cell lines (MCF-7 and MDA-MB-231) compared to the normal mammary epithelial cell line (MCF-10A), and expression by breast cancer tissue. We investigated the role of CXCR6 in breast cancer cell migration and invasion, which are key steps used by cancer cells to achieve metastatic goals. Breast cancer cells showed higher migratory and invasive potential towards the chemotactic gradients of CXCL16, which was significantly inhibited after CXCR6 blockade. Because our antibody array data showed phosphorylation of Src, FAK, and ERK1/2 in breast cancer cells following CXCL16 treatment, we used inhibitors against these molecules after CXCL16 treatment to determine the molecular mechanisms by which CXCR6 mediates migration and invasion. The results showed a significant decrease in migration and invasion, a reduction in F-actin polymerization, and decreased phosphorylation of Vav1, ezrin, cortactin, and paxillin. Taken together, this data suggests that Src, FAK, and ERK1/2 are required for CXCR6-mediated migration and invasion.
Cytokine-mediated regulation of CX3CL1 in human lung fibroblast: Involvement of STAT-1 signaling pathways (133.9)
Authors: Isozaki Takeo , Sato Michihito , Takahashi Ryo , Wakabayashi Kuninobu , Yajima Nobuyuki , Miwa Yusuke , Kasama Tsuyoshi
Published: January 2023

Introduction: CX3CL1 (fractalkine), a membrane-bound chemokine that induces both the adhesion and migration of leukocytes, is involved in the recruitment of cells into tissues undergoing inflammatory responses. To explore the regulation of CX3CL1 in pulmonary inflammation and fibrosis, CX3CL1 expression of by lung fibroblast was examined. Methods: Normal human lung fibroblasts were obtained from Promocell were incubated in the presence or absence of various inflammatory stimuli. Culture supernatants were collected, and soluble CX3CL1 levels were determined with an enzyme-linked immunosorbent assay. Expression of CX3CL1 mRNA transcripts in lung fibroblast was examined using quantitative TaqMan real-time polymerase chain reaction. Results: Interleukine (IL)-1β or interferon (IFN)-γ alone induced negligible soluble CX3CL1 secretion by human lung fibroblasts after 24 h. However, the combination of IL-1β and IFN-γ induced dramatic increases in both soluble CX3CL1 protein and mRNA transcripts in dose- and time-dependent manners. The secretion and expression of lung fibroblast-derived CX3CL1 were markedly reduced by specific inhibitors of the STAT-1 transcription factor. Conclusions: These findings suggest that lung fibroblasts are an important cellular source of CX3CL1, and may play roles in pulmonary inflammation and fibrosis like interstitial pneumonia which merges to rheumatoid arthritis and other autoimmune diseases.
Performance evaluation of two multiplex technologies for the measurement of serum cytokines in HIV-infected individuals (134.17)
Authors: Dabitao Djeneba , Margolick Joseph , Bream Jay
Published: January 2023

Simultaneous quantification of cytokines is a powerful tool to identify associations between host immune defense and human immunodeficiency virus (HIV) pathogenesis. None of the commercially available cytokine detection methods however, have been rigorously validated in the context of HIV infection. Here we compared performance characteristics of two multiplex assays: the Meso Scale Discovery (MSD) platform and the Cytometric Bead Array (CBA) in specimens obtained from HIV-infected subjects. Overall, the MSD had a wider dynamic range and lower limits of quantification which lead to the ability to detect endogenous levels of serum cytokines that were assumed to not be present by CBA. In recombinant cytokine-spiked sera, MSD had better precision and accuracy based on intra-assay variability less than 25% and percent recovery within 25% of added concentrations. Regardless of the quantitation range, we observed variability between the analytes in both platforms with respect to the capacity to recover a known amount of cytokine. Nonetheless, the MSD platform facilitates the reliable assessment of cytokines present in low concentrations in the serum of HIV-infected individuals.
IL-10 interruption of dendritic cell (DC) differentiation during the monocyte-to-DC transition is associated with increased levels of LC3, mature autophagosome formation, and survival of monocyte-macrophage-like cells. (134.21)
Authors: Santiago-Schwarz Frances , Valentino Alisa , Martin Carla , DiMaio Christopher
Published: January 2023

In immature DCs, fusion of autophagosomes containing Ag with MHC class II loading compartments (MIIC) has been linked to adaptive immunity. In monocyte (mono)-mϕs, fusion of autophagosomes with lysosomes yields autolysosomes and instead, innate immunity. Degradation of cargo (including apoptotic debris, leaky mitochondria) in autolysosomes may contribute to cell survival. Previously, we noted that despite IL-10-triggered apoptosis of newly committed DCs at the critical mono/DC interface, cells surviving such effects are non-antigen presenting mono-mϕ like cells avidly engaged in uptake of dying DCs. Here, we questioned if the surviving mono-mϕs exhibit increased autophagic activity. Flow cytometric analysis of mono-mϕs derived from human mono-DC precursors with DC growth factors +IL-10 revealed increases in the autophagy-associated LC3 protein vs. DC controls. Microscopic analysis of anti-LC3 stained cells showed increased punctate and ring-like patterns with IL-10, indicating formation of mature autophagosomes. Immunoblot analysis substantiated that cytosolic LC3I (18kd) was increased with IL-10 and converted to the autophagosome-associated LC3II (16kd) form. We advance the novel idea that autophagy occurs as a cytoprotective measure in concert with apoptotic stress triggered by IL-10. Aside from suppressing the onset of adaptive immunity by killing newly differentiated DCs, IL-10 may act to enhance innate immunity via pro-survival autophagic mechanisms.
Visualization of IL-15 expression in innate immune cells during homeostasis and inflammation (134.3)
Authors: Stoklasek Thomas , Colpitts Sara , Obar Joshua , Guo Caiying , Lefrançois Leo
Published: January 2023

IL-15 plays a multifaceted role in immune system homeostasis. To visualize IL-15 expression we created a transgenic mouse expressing emerald-GFP (EmGFP) under IL-15 promoter control. EmGFP expression was prevalent in dendritic cells (DC) and macrophages, but differed depending on DC subtype. Basophils, neutrophils, eosinophils, and mast cells expressed high EmGFP levels. Unexpectedly, NK cells and splenic γδ T cells also expressed EmGFP. Rapid upregulation of EmGFP after virus infection was dependent on both plasmacytoid DC and type I interferon, albeit to differing degrees. Microscopy revealed basal EmGFP expression by DC in lymphoid tissues that was greatly enhanced after virus infection. These results identified IL-15 as an innate cytokine, produced by multiple cell types during the early immune response to inflammation.
Interleukin-35 subunit dimerization and stability is unique among IL-12 cytokine family members (134.9)
Authors: Jones Lindsay , Collison Lauren , Vignali Dario
Published: January 2023

IL-35 is a newly discovered cytokine that contributes to the suppressive capacity of regulatory T cells. IL-35 is a heterodimer consisting of an α chain (p35) and a β chain (Ebi3). It is a member of the IL-12 cytokine family, which is characterized by the sharing of three α (p19, p28, p35) and two β (p40 and Ebi3) subunits, and also includes IL-12 (p35/p40), IL-23 (p19/p40) and IL-27 (p28/Ebi3). In this study, relative pairing and secretion efficiencies and the dimerization interfaces of IL-12 family members were characterized, with emphasis on IL-35. Specific p35 residues that are known to contribute to formation of the IL-12 interface were assessed for their contribution to the IL-35 interface in mutagenesis studies. Similarly, several candidate Ebi3 residues were screened for their contribution to both IL-27 and IL-35 interfaces. Several residues were identified as critical to the IL-12 or IL-27 interfaces. Conversely, no single mutation was identified that completely disrupts p35/Ebi3 pairing, indicating that p35 and Ebi3 may pair differently to form IL-35. Preliminary data also indicate that IL-35 is poorly secreted by transfected cells, compared to IL-12 and IL-27, suggesting IL-35 formation is unfavorable or the secreted protein in unstable. Thus IL-35 appears to be different from other IL-12 family members in terms of both dimerization interface and stability. This work is supported by grants from the NIH and ALSAC.
Pathogenic or non-pathogenic subpopulations of Th17 cells are generated by stimulation of naïve CD4 cells via antigen/APC or anti-CD3/CD28 antibodies (135.10)
Authors: Shi Guangpu , Vistica Barbara , Lovaas Jenna , Tang Cuiyan , Aziz Mehak , Gery Igal
Published: January 2023

Th17 cells play a major role in immunopathogenic processes. McGeachy et al, (Nat. Immunol., 2007) showed that two distinct subsets of Th17 were generated from activated CD4 cells when different polarization modes were used, with only one subset being encephalitogenic. Here, we analyzed the biological features of Th17 generated by the same polarization mechanism, i.e., incubation with IL-6 and TGF-b, but by two different modes of stimulation. Naïve CD4 cells, collected from TCR transgenic mice specific against hen egg lysozyme (HEL), were activated by either HEL and APC (“HA”), or by plate-bound antibodies against CD3/CD28 (“PbAb”). PbAb Th17 produced moderately higher levels of IL-17 than HA Th17, but the latter cells released profoundly higher levels of IL-22. Interestingly, Th17 cells of the two subsets produced similar levels of IL-10, shown by both analysis of the culture supernatants and intracellular staining. Importantly, when adoptively transferred into recipients expressing HEL in their lens, only HA Th17 cells induced ocular inflammation in recipients, while no disease was detected in eyes of PbAb cell recipients. It is also of note that the two Th17 subsets differed in their profile of chemokine transcripts, resembling the two different profiles recorded by McGeachy et al with the pathogenic and non-pathogenic subsets. Thus, the pathogenic or non-pathogenic Th17 subsets are generated in our system, without pre-activation, by two stimulation modes.
Mast cells influence disease severity in a murine model of relapsing-remitting multiple sclerosis. (135.15)
Authors: Walker Margaret , Amir-Sayed Blayne , Brown Melissa
Published: January 2023

Although mast cells (MCs) are critical mediators of allergic and innate immune responses, they also modulate adaptive immune responses. Utilizing WBB6c-kitW/Wv mast cell-deficient mice, MCs have been implicated in the promotion of disease severity in several murine models of human autoimmune disease. WBB6c-kitW/Wv mice have two independent mutations that profoundly inhibit MC development. We previously demonstrated that MCs influence disease severity in a murine model of chronic-progressive multiple sclerosis (CP-MS), specifically by influencing cellular trafficking into the CNS and blood brain barrier permeability. However, this model does not mimic the most common form of the human disease, relapsing-remitting MS (RR-MS). Therefore, to test the effect of a MC deficiency on a RR form of disease, we transferred the c-kitW/Wv mutations onto the SJL background, a strain that is susceptible to RR disease. These animals are MC-deficient in all tissues examined, carry mild hematopoietic deficits, but have relatively normal thymic T cell compartments. SJLc-kitW/Wv mice display significantly decreased cumulative EAE disease scores and a delay in the day of peak acute disease compared to their wild type littermates. These differences in disease phenotypes are abrogated upon reconstitution of wild type bone marrow-derived MCs. Thus, MCs also influence disease severity in a RR murine model of MS, a finding with potential clinical relevance for the heterogeneous MS patient population.
Effect of Kv1.3 inhibition on T cell development, activation and signaling (135.23)
Authors: Rosenzweig Jason , Lebson Lori , Mancini Justin , Calabresi Peter , Whartenby Katharine
Published: January 2023

The voltage-gated potassium channel Kv1.3 is highly expressed in effector memory T cells (TEM) but not in central memory (TCM ) or naïve T cells. TEM are pathogenic in several autoimmune diseases. Acute blockade of Kv1.3 by the sea anemone toxin ShK reduces activation induced proliferation of TEM. However, in Kv1.3-/- mice, in which the gene was absent from development, no immune system defects were found, which was attributed to compensatory channels (Koni et al. JBC, 2003). The limitations of pharmacological blockade and gene-targeted deletion led us to develop a lentiviral system to co-express a dominant negative Kv1.3 and GFP. We have been using this lentivirus to query the role of Kv1.3 both in the development and activation of T cells. We have conducted studies in which we have suppressed channel expression in the development of T cells from a gene-modified bone marrow transplant and also assessed the impact of blocking channel expression during antigen-specific activation of CD4+ T cells. Using the antigen specific mouse system allows multiple advantages over previous studies including the feature that specific T cells can be tracked and monitored and compared to unmodified T cells. Initial results showed that Kv1.3-silenced CD4+ T cells skewed towards the TCM phenotype. We will report on our additional results using this lentiviral expression system, which is allowing us to study the role of Kv1.3 in T cells in mice and its effect on signaling pathways.
Memory B cells from relapsing remitting multiple sclerosis patients elicit functional responses by CD4+ T cells in response to neuro-antigens (135.24)
Authors: Monson Nancy , Harp Christopher , Ireland Sara , Davis Laurie , Cassidy Bonnie , Cravens Petra , Stuve Olaf , Lovett-Racke Amy , Eagar Todd , Greenberg Benjamin , Racke Michael , Cowell Lindsay , Karandikar Nitin , Frohman Elliot
Published: January 2023

Recent evidence suggests that B and T cell interactions may be paramount in relapsing remitting multiple sclerosis (RRMS) disease pathogenesis. We hypothesized that memory B cell pools from RRMS patients may specifically harbor a subset of potent neuro-antigen presenting cells that support neuro-antigen reactive T cell expansion and activation. To assess the potential influence of neuro-antigen specific memory B cells from RRMS patients on autologous CD4+ T cell activation, we compared CD80 and HLA-DR expression, IL-10 and LTα secretion, neuro-antigen binding capacity, and functional neuro-antigen presentation by memory B cells from RRMS patients and compared these parameters to naïve B cells from RRMS patients and to memory and naïve B cells from healthy donors (HDs). We identified memory B cells from a subset of RRMS patients that were functionally capable of presenting neuro-antigen to autologous T cells. Notwithstanding the fact that the phenotypic parameters that promote efficient antigen presentation were observed to be similar between RRMS and HDs memory B cells, a corresponding capability to present neuro-antigen was not observed in HD memory B cells. Our results demonstrate for the first time that the memory B cell pool in RRMS harbors neuro-antigen specific clones that can activate T cells.