bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-10 produced by host animals, but not by transduced donor B cells, is required for tolerance delivered by B-cell gene therapy (136.34)
Authors: Su Yan , Noben-Trauth Nancy , Scott David W.
Published: January 2023

Our previous studies indicated that activated B cells, which were retrovirally transduced with an IgG-antigen fusion construct, could effectively induce antigen-specific tolerance in vivo. Since IL-10 is an important cytokine for maintaining immune tolerance and suppression, in this study we investigated the role of IL-10 in the B-cell delivered gene therapy for tolerance induction. First we transduced either wild type (wt) or IL-10 knockout (KO) B cells with ovalbumin (OVA) peptide-IgG fusion protein via a retroviral vector and found that both wt and IL-10 KO B cells are tolerogenic antigen-presenting cells (APC) capable of inducing antigen-specific tolerance in host animals.. Next we tested whether IL-10 produced by host animals is required for tolerance induction. Animals that received OVA peptide-IgG B cells were injected weekly with anti-IL-10 receptor antibody (1B1.3a) or a control antibody (rat IgG1 GL113). This in vivo anti-IL-10 receptor blockade completely abrogated the tolerogenic effects of OVA peptide-IgG B cells. Together, our results suggest that tolerance induced by B cells requires the IL-10 produced by host, but not by donor B cells. These data suggest that regulatory T cells which produce IL-10 are involved in the tolerance induction by this B-cell gene therapy protocol. (Supported by NIH grants RO1 AI035622 and DK068343)
Th17 cells mediate sustained autoimmune inflammation and are highly resistant to restimulation-induced cell death (137.17)
Authors: Shi Guangpu , Ramaswamy Madhu , Vistica Barbara P. , Tan Cuiyan , Wawrousek Eric F. , Siegel Richard M. , Gery Igal
Published: January 2023

Both Th1 and Th17 T-cell subsets can mediate inflammation, but the kinetics of the pathogenic process mediated by these two subsets has not been investigated. Using an experimental system in which TCR-transgenic Th1 or Th17 cells specific for hen egg lysozyme (HEL) induce ocular inflammation in recipient mice expressing eye-restricted HEL, we found important differences in the in vivo behavior of these two subsets. Th1 cells initially proliferated considerably faster and invaded the eye more quickly than their Th17 counterparts, but then disappeared rapidly. By contrast, Th17 cells accumulated and remained the majority of the infiltrating CD4 cells in the eye for at least 15 days after transfer, mediating more long-lasting pathological changes. Th17 cells were highly resistant to restimulation-induced apoptosis, a major pathway by which autoimmune and chronically restimulated Th1 cells are eliminated. Th17 cells had reduced FasL production and resistance to Fas-induced apoptosis relative to Th1 cells despite normal surface expression of Fas. Moreover, Th1 recipient eyes had a higher expression level of FasL than Th17 recipient eyes. Resistance to programmed cell death in Th17 cells may enable more long-lasting responses against pathogens, but may also enable the chronic autoimmune tissue destruction that has been ascribed to Th17 cells.
Gender-dependent differential antigen uptake by CD11bloF4/80lo macrophages from experimental autoimmune encephalomyelitis resistant male and susceptible female SJL mice (137.2)
Authors: Hussain Shabbir , Stohlman Stephen A
Published: January 2023

Macrophages (MΦ) preferentially regulate induction of antigen-specific Th1 and Th2 immune responses to protein antigens in female and male SJL mice, respectively. Alexa Flour 488 conjugated ovalbumin (OVA) and self-quenched DQ-OVA that fluoresces following lysosomal degradation, were used to determine gender-dependent differences in antigen uptake and processing in peritoneal MΦ. Antigen uptake and processing are higher in CD11bhiF4/80hi compared to the CD11bloF4/80lo MΦ of both genders. CD11bhiF4/80hi MΦ in both genders exhibit similar levels of antigen uptake, processing and MHC class II expression. By contrast, CD11bloF4/80lo MΦ from males show increased antigen uptake and decreased MHC Class II expression compared to female CD11bloF4/80lo MΦ. Despite differences in antigen uptake and MHC class II expression, CD11bloF4/80lo MΦ from both genders show similar kinetics of antigen processing. CD11bloF4/80lo MΦ from both genders presented antigen to CD4+ T cells; however, increased IL-10 was induced during T cell activation by male CD11bloF4/80lo MΦ compared to female CD11bloF4/80lo MΦ. These data demonstrate increased antigen uptake, diminished Class II expression and preferential IL-10 secretion by CD11bloF4/80lo MΦ derived from Th2 responsive EAE resistant male SJL mice. This work was supported by National Institute of Health grant NS53824.
Human keratinocytes release and respond to danger signals in cutaneous lupus erythematosus (137.33)
Authors: Wittmann Miriam , Wang Dong , Eiz-Vesper Britta , Werfel Thomas
Published: January 2023

Lupus erythematosus (LE) is a chronic multisystem disease and cutaneous manifestations belong to the most common clinical features. It was the aim of this study to further decipher the role of keratinocytes in autoinflammation. In immunohistochemistry we found a high expression of inducible HSP70, IL-18R and IL-18 in lesional LE skin. Increased expression of TNFalpha and high mobility group box-1 (HMGB-1) has been described previously. Studies with cultured, patient derived keratinocytes showed that IL-18 stimulation results in significant TNFalpha production in LE derived but not in healthy keratinocytes. Furthermore, we could show that keratinocytes release high levels of HSP70 into the supernatant which was not due to apoptosis. Here we provide evidence, that HSP70-peptide complexes are internalised by keratinocytes and that antigenic peptides induce the production of IFNgamma in T cells via this route. Of note, HMGB-1 as well as TNFalpha significantly enhanced the uptake of HSP70 into human primary keratinocytes. In conclusion, we provide evidence that skin resident cells play an active part in maintaining LE inflammation also by means of secretion of molecules which belong to the danger-associated molecular pattern (DAMP) family. This study was supported by DFG grant Wi1822/5-1 and SFB566/A6.
Residual T cell potential in common myeloid progenitors (138.14)
Authors: Chi Anthony WS , Bhandoola Avinash
Published: January 2023

Downstream of hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs), common myeloid progenitors (CMPs) are thought to represent a progenitor population that contributes to the development of myeloid and erythroid cells. CMPs do not contribute to T cell development in mice, as upon intrathymic transfer they fail to generate T cells in the thymus. However, when co-cultured on OP9 stromal cells expressing Delta-like Notch ligands, CMPs generated T cells, though less efficiently than HSCs or MPPs. Such inefficiency was demonstrated by the increased sensitivity of CMPs to GSI blockade. This defect correlated with reduced expression of Notch1 and also Lunatic fringe. Consistent with this finding, overexpression of ICN1 to mimic constitutively active Notch1 signaling was sufficient to drive CMPs into the T lineage developmental pathway in vivo, and resulted in development of T cell leukemia. This study shows CMPs are still receptive to Notch1 signaling and Notch-mediated T cell leukemogenesis. We are currently exploring mechanisms that might signal downregulation of Notch1 and Lunatic fringe in CMPs, and so initially constrain T potential. We are also reconstituting expression of Notch1 and Lunatic fringe in CMPs using retroviruses, and will determine whether such manipulations are sufficient to confer T potential to CMPs in vivo, or whether additional mechanisms that constrain T potential exist.
Characterizing Thymocyte Migration in the Medulla Using Two-Photon Microscopy (138.20)
Authors: Ladi Ena , Le Borgne Marie , Liao Ying Fang , Robey Ellen
Published: January 2023

To establish tolerance, a developing thymocyte must be screened for self-reactivity before exiting the thymus. However, the mechanism by which a thymocyte searches the medulla and antigen-presenting cells for self-antigens is not clear. We have developed a cut thymic lobe preparation that allows us to document thymocyte migration within the medulla by two-photon laser scanning microscopy. Medullary thymocytes migrate rapidly through confined random walk making short and dynamic contacts with dendritic cells. In response to negative selection signals, thymocyte migration is significantly slower and constrained to smaller areas. In addition, self-reactive thymocytes engage in long lasting contacts with dendritic cells. Polyclonal medullary thymocytes consist of a small but significant population of relatively slow thymocytes with more constrained migration that may correspond to auto-reactive thymocytes undergoing negative selection. Taken together, our data defines scanning strategies used by medullary thymocytes during negative selection and suggests that a significant proportion of medullary thymocytes are actively receiving negative selecting signals.
Egr1-Rcan1 axis serves as a molecular switch from activation to inhibition in IgE-mediated signalling (139.3)
Authors: Lin Tong-Jun , Yang Yong Jun , Molkentin Jeffery D , Berman Jason N
Published: January 2023

Mast cells play a central role in allergy. Aggregation of FcεRI activates a cascade of signaling events leading to mast cell activation. Subsequently, inhibitory signals are switched on for turning off activated signals. Here we identified that Rcan1 serves as a negative regulator for turning off FcεRI-mediated mast cell activation. FcεRI-induced Rcan1 expression was identified by suppression subtractive hybridization and verified by real-time quantitative PCR and Western blotting. Deficiency of Rcan1 led to increased calcineurin activity, increased NFAT and NF-κB activation, increased cytokine production and enhanced IgE-mediated late phase cutaneous reactions. Forced expression of Rcan1 in wild type or Rcan1 deficient mast cells reduced FcεRI-mediated cytokine production in vitro. Rcan1 deficiency also led to increased FcεRI-mediated mast cell degranulation and enhanced passive cutaneous anaphylaxis. Analysis of the Rcan1 promoter identified a functional Egr1 binding site. Rcan1 expression is preceded by Egr1 expression. Egr1 is a positive regulator of FcεRI-induced cytokine production. Biochemical and genetic evidence suggested that Egr1 controls Rcan1 expression. Our results identified Rcan1 as a novel inhibitory signal in FcεRI-induced mast cell activation and established a new link of Egr1 and Rcan1 in FcεRI signaling.
Lack of negative selection of CD4 T cells by bone marrow (BM)-derived cells causes fatal autoimmunity manifested as acute GVHD (141.16)
Authors: Chen Leo Cheng , Guo Xiaohua , Jensen Peter E , Chen Xinjian
Published: January 2023

While it is well established that BM-derived APCs play an essential role in the induction of self-tolerance, the biological consequence of losing this tolerogenic mechanism has yet to be determined. We generated BM chimeras by transplanting BM from MHC II-/- B6 mice into lethally irradiated Rag-/- or WT B6 mice. Lymphopoesis in the recipient mice initially appeared normal, but 5 wks post BMT, most mice became rapidly ill and died shortly afterwards. Histological examination revealed extensive epithelial apoptosis of the GI tract and skin on a pauci-inflammatory background. The liver showed marked vasculitis. The thymus underwent rapid atrophy and loss of developing thymocytes. The heart, lung, kidney, brain, salivary glands, pancreas, pancreatic islets and gonads were not affected. CD4 T cells in the thymus reacted vigorously against B6 DCs in vitro. Although thymic selection of CD8 T cells was unaltered, the CD8 T cells in the lymph nodes of the diseased animals were activated. Transfer of spleen cells from the diseased animals into Rag-/- mice resulted in similar tissue damage and accelerated death of recipients, but no disease if the Rag-/- mice were MHC II-/-. Further experiments involving MHC II-/- BM to MHC I-/- chimeras demonstrated that expression of MHC I by host tissue is also required for disease development. Our findings demonstrate that a consequence of lack of negative selection of CD4 T cells by BM-derived cells is fatal autoimmunity manifested distinctively as acute GVHD. The results also suggest that aGVHD can be induced solely by CD4 T cells generated in the recipients if the donor BM cells lack the MHC II type expressed by thymic epithelial cells of the recipients.
Rare dual TCR T cells contribute substantially to alloreactivity (141.2)
Authors: Morris Gerald P , Allen Paul M
Published: January 2023

T cell recognition of allogeneic MHC has long been recognized, though the nature of the alloreactive TCR repertoire is unknown. We hypothesize that the 1-3% of T cells expressing dual TCR due to incomplete allelic exclusion of TCRα are highly alloreactive due to non-thymically selected secondary TCR. Using the 4 available antibodies against TCRVα we observe an increased frequency of dual TCR T cells among alloreactive T cells in vitro. We quantitatively assessed the contribution of secondary TCR to the alloreactive repertoire by ELISPOT analysis; TCRA-C mice, incapable of expressing secondary TCRα, have a 50% decrease in alloreactive precursor frequency indicating secondary TCR contribute disproportionately to the alloreactive repertoire. We directly demonstrate expansion of the alloreactive repertoire by identification of a secondary non-thymically selected TCR with specific alloreactivity distinct from the primary TCR. The importance of dual TCR T cells is underscored by the dramatic increase of dual TCR among T cells mediating parent to irradiated F1 graft-vs.-host disease (GvHD); dual TCR comprised as much as 40% of peripheral T cells in mice with GvHD, compared to 3% observed in control mice. Together these studies convincingly demonstrate the importance of dual TCR T cells in alloreactivity. NIH AI061173 (PMA) and W.M. Keck Foundation Fellowship in Molecular Medicine (GPM).
The effect of the αGal epitope in the response to ECM in a nonhuman primate model (141.26)
Authors: Daly Kerry A , Stewart-Akers Ann A , Cordero Kevin , Johnson Scott A , Badylak Stephen F
Published: January 2023

The Galα1,3Galβ1,4GlcNAc-R (Gal) Epitope is a major factor in the hyperacute rejection of pig organs transplants in primates. Biologic scaffold materials used for tissue reconstruction and composed of xenogeneic extracellular matrix (ECM) may contain the Gal epitope. However, the effect this epitope has upon the host response is controversial. The present study investigated the effect of the Gal epitope upon the host response to a porcine-derived ECM in an African Green monkey (Cholrocaebus aethiops) abdominal wall resection model. Histologic methods, serology, complement-dependent cytotoxicity (CDC) and gene expression profiling were used to evaluate the host response to allogeneic and both wild-type and Gal-deficient xenogeneic scaffold materials. Although expression of the Gal epitope induced an increase in serum anti-Gal antibodies in recipients, no other differences were noted in the host response between test articles. All ECM scaffolds were well tolerated and showed constructive remodeling during the test period. Recipients of all test articles showed no histologic or humoral evidence of sensitization when a second scaffold was implanted 45 days after the original surgery. The findings of the present study show that the Gal epitope within porcine-derived ECM scaffold material causes a serum antibody response, but no adverse effect upon tissue remodeling.
The role of DAMPs in the host immune and remodeling response to xenogeneic biologic scaffold materials (141.27)
Authors: Brown Bryan N. , Daly Kerry A. , Lotze Michael T. , Badylak Stephen F.
Published: January 2023

Biologic scaffolds composed of extracellular matrix (ECM) have been used in pre-clinical and clinical applications with few reports of adverse immunologic reactions. Following implantation, ECM scaffolds are readily remodeled by the host; however, the mechanisms underlying the host immune and remodeling responses are not fully understood. A variety of molecules within ECM scaffolds, as well as their degradation products, have been shown to influence the host response. Damage associated molecular pattern molecules (DAMPs) are released during non-apoptotic cell death, and may act as either pro- or anti-inflammatory mediators. Consequently, the presence of DAMPs within a biologic scaffold material may direct the host immune and remodeling responses following implantation. In the present study, the presence of several well-established DAMPs within ECM scaffold materials was evaluated. In addition, the presence of DNA and the redox state of the scaffold microenvironment, which may influence the inflammatory potential of DAMPs, were assessed to determine their effects upon host immune and remodeling responses. The results of the present study demonstrate the complex nature of the interactions between the host immune response, local microenvironment, and tissue remodeling, following the implantation of biologic scaffold materials.
WIP peptide stabilizes WASP in cells from Wiskott-Aldrich syndrome (WAS) patients with mutations in the WIP-binding domain (33.16)
Authors: Massaad Michel , Giliani Silvia , Notarangelo Luigi , Geha Raif S.
Published: January 2023

WAS is an X-linked immunodeficiency characterized by eczema, thrombocytopenia and recurrent infections. WAS-protein (WASP) links cell surface signals to the actin cytoskeleton. WASP is important in T cells for IL-2 production, proliferation, and migration. The WAS-interacting protein (WIP) stabilizes WASP. WASP level is severely reduced in T cells of WIP KO mice. The majority of missense mutations in WAS patients are in the WIP binding domain (WBD) of WASP and interfere with WIP binding, resulting in low WASP levels.

We identified a 35 a.a. fragment of WIP (termed nWIP), which when fused to the C-terminal of EGFP (EGFP-nWIP), interacts with WASP in Jurkat cells. When introduced into WIP KO T cells, EGFP-nWIP increased the level of WASP to normal, as determined by FACS analysis. EBV-transformed B cell lines from WAS patients with mutations in the WBD of WASP expressed low levels of WASP, but had normal mRNA levels. Introduction of EGFP-nWIP into these lines resulted in increased levels of WASP, up to normal. Two cell lines from patients with WAS mutations outside the WIP-binding domain did not show an increase of WASP after introduction of EGFP-nWIP.

Our results demonstrate that nWIP stabilizes WASP and ameliorates WASP levels in B cells from WAS patients with missesense mutations in the WBD of WASP. We will determine if nWIP will correct the functional defects in cells from these patients. (Supported by USPHS grant HL059561 and the PERKIN fund.)
CXCR6/CXCL16 is required for optimal NKT cell and dendritic cell interactions (33.2)
Authors: Veinotte Linnea Lora , Johnston Brent
Published: January 2023

Natural killer T (NKT) cells are a conserved T cell sublineage which rapidly produces cytokines upon activation by glycolipid antigens (i.e. α-GalCer) presented by dendritic cells (DC). Cross-talk between NKT cells and DCs is important in the initiation of anti-tumour responses. The objective of this study was to determine the role of CXCR6/CXCL16 in NKT cell-DC interactions. The chemokine receptor, CXCR6, is highly expressed on NKT cells, while its ligand, CXCL16, is expressed on DCs. When liver NKT cells were cultured with recombinant CXCL16 in vitro they were not activated directly. However, CXCL16 did enhance IFN-γ production in vitro when NKT cells were stimulated with suboptimal doses of plate bound anti-CD3. The presence of CXCL16 on the surface of DC is also important for NKT cell production of IFN-γ. NKT cells cultured with α-GalCer loaded CXCL16+ DC produced more IFN-γ than those cultured with CXCL16- DC. CXCL16+ DC stimulated more NKT cell IFN-γ production than CXCL16- DC in vivo as well. CD8+ DC constitutively express CXCL16 while all other DC subsets upregulate CXCL16 expression following α-GalCer stimulation. This is dependent on NKT cells since CXCL16 upregulation did not occur in NKT cell deficient (Jα18-/-) mice. DC maturation is also attenuated in the absence of CXCL16 signaling. These results suggest that CXCR6/CXCL16 is required for full NKT cell and dendritic cell activation/maturation.
Orchestration of B cell function by the ERM protein ezrin (34.2)
Authors: Gupta Neetu , Bagheri Nayer , Parameswaran Neetha
Published: January 2023

Appropriate B cell responses hinge on finely balanced signal transduction pathways as well as regulated membrane remodeling. Membrane remodeling requires its uncoupling from the underlying cortical cytoskeleton but its regulation and impact on B cell function is poorly understood. We have identified ezrin, a member of the ezrin-radixin-moesin (ERM) family, as a regulator of membrane-cytoskeletal uncoupling in response to both B cell antigen receptor (BCR) and chemokine stimulation. Both stimuli induce dephosphorylation of ezrin on amino acid T567 in the actin-binding domain of ezrin, resulting in conformational inactivation of ezrin. Expression of a mutant of ezrin (T567D) that constitutively links the membrane to the actin cytoskeleton interferes with BCR-induced lipid raft coalescence, and SDF-1-dependent B cell migration. Our data support a critical role for T567 dephosphorylation in the early membrane remodeling events associated with antigen and chemokine signaling. Mice with a conditional deletion of ezrin in the B cell lineage exhibit defects in peripheral B cell homeostasis. Ezrin-deficient B cells from these mice show impaired in vitro migration and in vivo homing. Our data suggest that ezrin has the potential to shape activation and migration associated with B cell homeostasis and immune response.

Supported by grants from NIH (DK068292), Cancer Research Institute, Department of Defense.
Dual function for a vision-related molecule: retinoic acid in the eye may contribute to ocular immune privilege (39.18)
Authors: Caspi Rachel R , Zhou Ru
Published: January 2023

Ocular immune privilege is a complex phenomenon that encompasses both local and systemic mechanisms. Ocular fluids contain TGFβ as well as several neuropeptides that are thought to contribute to ocular immune privilege. Retinoic acid (RA) and its metabolites are abundant in the eye, as they constitute an integral part of the chemistry of vision. RA was recently shown to be involved in immune regulation at mucosal surfaces but its role, if any, in immune responses involving the eye has not been studied. We therefore examined the ocular fluids for immunoregulatory activity that could be attributed to RA. Confocal and real-time RT-PCR analyses showed expression of RA-synthesizing enzymes RALDH 1, 2 and 3 in the cornea and retina. Aqueous humor (AH) added to T cells undergoing Th1 or Th17 polarization inhibited acquisition of effector function as determined by intracellular staining for IFNγ and IL17, and instead caused their massive conversion into FoxP3+ Tregs. This involved TGFβ-driven upregulation of RARα and could be reversed by antagonizing either RA or TGFβ using LE540 or anti-TGFβ antibodies. In addition, AH-conditioned dendritic cells had reduced ability to support acquisition of effector function by T cells that had not themselves been exposed to AH. Our data support the notion that the role of RA in the eye is not limited to the visual process but also contributes to ocular immune privilege.

NIH intramural funding
PI3Kgamma regulates mucosal immune responses to epicutaneous immunization with cholera toxin as adjuvant (39.2)
Authors: Papenfuss Tracey  L , Satoskar Abhay , Lu Bao , Boyaka Prosper N
Published: January 2023

PI3Kgamma is a phosphoinositide3-kinase predominantly expressed in leucocytes and activated by G-protein-coupled receptors. Cholera toxin (CT) is an AB5 enterotoxin that binds to GM1 ganglioside and catalyzes ADP-ribosylation of Gsalpha. CT is also an effective adjuvant that induces systemic and mucosal immunity. We examined the role played by PI3Kgamma in the mucosal adjuvant activity of CT for epicutaneous vaccines. PI3Kgamma KO and control C57BL/6 mice were immunized three times by transcutaneous application of 50 micrograms of CT with 1 mg ovalbumin (OVA). Control mice developed significant levels of OVA-specific IgA Abs in the serum and in fecal extracts. The adjuvant activity of CT in control mice also resulted in high OVA-specific IgG responses in the serum with IgG1>IgG2b>>IgG2a. Interestingly, the magnitude of OVA-specific serum IgG responses and fecal IgA Abs, were significantly enhanced in PI3Kgamma KO mice. The lack of PI3Kgamma signaling also enhanced Ab responses to CT itself, but did not affect the Th1/Th2 profile of responses. These results suggest that PI3Kgamma plays a regulatory role on immune responses to epicutaneous vaccines with ADP ribosyl-transferase adjuvants.
Characterization of antibody responses to soy proteins following oral vaccination with adjuvanted soymilk formulations (39.39)
Authors: Oakes Judy L. , Piller Kenneth J. , Bost Kenneth L.
Published: January 2023

We are exploring a novel platform technology using soybean seed-based formulations for the development of subunit vaccines that can be delivered via an oral route. One requirement for an effective subunit vaccine formulation is the inclusion of an adjuvant in order to elicit a robust immune response within the intestinal mucosa. When considering a soybean seed based formulation it is critical that there is no immune response to native soy proteins when administered orally with an adjuvant. One of the many advantages of a soybean seed-based platform for vaccine development is the fact that virtually every animal and human has been exposed to soy proteins given that soy is a major component of almost every diet. We hypothesize that routine consumption of soy proteins in a diet should effectively tolerize against soybean proteins and therefore abrogate the adjuvant effect of soy proteins. To test this hypothesis, we orally vaccinated groups of mice with formulations containing high (15mg) and low (1mg) doses of soybean seed extracts, along with cholera toxin (CT) as an adjuvant. As expected, there were no detectable sera IgE or IgG response in either group of vaccinated mice. Furthermore, there was no detectable IgA response against soy proteins in fecal extracts of immunized animals. Immune responses to CT in all groups were as predicted with a robust IgG response reflecting a Th2 subtype and an IgA response in fecal extract. Collectively, these data demonstrate that adjuvanted soybean-based formulations do not induce immune responses to soybean seed proteins, and underscore the vast potential for a novel soybean-based platform technology that can provide safe, stable and cost-effective vaccines worldwide.
Differential effects of mucosal adjuvant cholera toxin (CT) on macrophage cyclooxygenase-2 (COX-2) and endoplasmic reticulum (ER) stress (39.44)
Authors: Kogiso Mari , Pantuso Traci , Henriksen Ruth Ann , Dorey C. Kathleen , Shibata Yoshimi
Published: January 2023

CT is known to induce enhanced COX-2 activity and increased release of prostaglandin E2 (PGE2) in macrophages (MØ) and mucosal cells; CT also induces ER stress and unfolded protein responses. Since CT alone does not induce COX-2 transcription in MØ, post-transcriptional mechanisms up-regulating COX-2 appear essential, but remain elusive. To determine whether CT-induced ER stress is associated with regulation of MØ COX-2, RAW 264.7 MØ were treated overnight with CT (0.2 and 1 µg/ml) followed by 6 h treatment with heat-killed Mycobacterium bovis bacillus Calmette-Guérin (BCG, 5, 50, and 100 µg/ml). We found that pre-treatment with CT caused dose-dependent inhibition of BCG-induced COX-2 expression and marked elevation of growth arrest- and DNA damage-inducible gene 153 (GADD153), suggesting increased ER stress. Expression of glucose-regulated protein 78 (GRP78), an ER chaperone, was unchanged. In sharp contrast, if MØ had been pre-treated with BCG overnight before 6 h CT treatment, COX-2 expression was enhanced, GADD153 was not induced, and GRP78 expression was unchanged. Either pre- or post-treatment with CT resulted in inhibition of BCG-induced TNFα production. Our results indicate that the differential COX-2 regulation by CT is dependent on an ER stress/GADD153 pathway. Further studies will be conducted to assess whether the ER stress-dependent biphasic COX-2 expression is also observed in mucosal MØ and epithelial cells in CT-treated animals. (Supported by NIH HL71711, DOD DAMD 17-03-1-0004, BCCRP 06BS-04-9615)
The role of FasL and IFNγ in a model of AAV-mediated chronic liver injury in the mouse (39.52)
Authors: Spahn Jessica , Azadniv Mitra , Clementi Alicia , Crispe I. Nicholas
Published: January 2023

As models of chronic liver injury in the mouse are limited, we have developed such a model in the C57B6 mouse that utilizes Adeno-associated viral vectors (AAV) engineered to express both enhanced Green Fluorescent Protein and the CD8+ epitope SIINFEKL. After injection of this vector directly into the liver, 1x104 SIINFEKL-specific OT-1 cells are administered. Injection of these cells results in an increase in OT-1 cell numbers in the liver until Day 20 correlating with liver damage from Days 10-25. Addition of these cells does not cause decreased vector DNA but significantly decreases vector mRNA expression. To define the mechanisms by which OT-1 cells mediate their effect, we focused on both IFNγ and Fas. In looking at the role of these two molecules, we have found that IFNγ is important in the activation and/or recruitment of the OT-1 cells as IFNγR KO hosts have a significant decrease in the number of OT-1 in the liver at Day 20 compared to their WT controls. The same has been found when comparing WT and Faslpr hosts indicating a role of FasL in the same activation or recruitment processes. FasL has been shown by Suzuki et al to play a role in costimulation in a peptide model, but this phenomenon has not been seen in other viral or vector models1. The role of these two molecules in our AAV model may give clues to the molecular events that occur in other types of hepatitis.

Suzuki I JI 2000 Nov 15; 165(10):5537-43.

Support: NIH RO1 AI064463
ICAM-1 mediated NK cytotoxicity is enhanced by thymosin beta 4 (40.12)
Authors: Lee Ha-reum , Cho Dae-Ho
Published: January 2023

Thymosin beta 4, which is the major G-actin sequestering protein, has been shown to have ubiquitous distribution and multiple biological activities. However, thymosin beta 4's functions in relation to natural killer (NK) cells are still unknown. In this study, we show that synthetic thymosin beta 4 peptide increases NK cell cytotoxicity in a dose-dependent manner on human NK cell line, NK-92MI. Addition of thymosin beta 4 enhances NK cell aggregation and intercellular adhesion molecule-1 (ICAM-1) surface expression that are involved in NK cell cytotoxicity. Neutralizing ICAM-1 activity impairs thymosin beta 4-enhanced NK cell cytotoxicity. Furthermore, thymosin beta 4 stimulates degranulation of cytolytic granules, which is verified by CD107a assay, and up-regulates NK cell cytotocixity on primary human NK cells. Our findings revealed that thymosin beta 4 enhances NK cell cytotoxicity mediated by ICAM-1 through secretion of cytolytic granules to target cells. This suggests that thymosin beta 4 is a key activator of NK cell cytotoxicity.
Increased peripheral natural Tregs in prostate cancer patients is correlated with PSA (40.13)
Authors: Koksoy Sadi , Cicek Nilüfer , Akin Yigit , Erdogru Tibet , Baykara Mehmet , Ciftcioglu Mehmet Akif , Yucel Selcuk
Published: January 2023

The aims of this study were to analyze peripheral natural regulatory T cells (Treg) in Prostate Cancer (PCa) patients, regarding density and correlation with serum Prostate Specific Antigen (PSA). Peripheral Tregs from 75 subjects undergoing diagnostic prostate biopsies were enumerated by two sets of markers, CD4+CD25highFoxp3+ and CD4+CD25+CD127low/-. Association between peripheral Treg and serum PSA values was first determined in the entire population, including people with no prostate pathology (healthy) , PCa and Benign Prostate Hyperplasia (BPH) patients, and second, in 9 PCa patients before and after curative prostatectomy. Peripheral Treg frequencies were significantly increased in PCa patients (n=30) compared with BPH patients (n=30) and healthy subjects (n=15) by both methods (p<0.01). The percentage of Treg in BPH patients was also slightly but significantly higher than that of healthy subjects. Importantly, the increase paralleled the elevation in total serum PSA levels, demonstrating a strong positive correlation (r =0.70 by both methods). Additionally, prostatectomy concomitantly restored both peripheral Treg numbers and serum PSA levels. In conclusion, peripheral Treg densities are not only increased in PCa and BPH patients but are also strongly correlated with PSA, suggesting that PSA may have a role in induction and/or maintenance of increased proportion of Tregs in these people.
Immunologic mechanisms against an SV40 viral oncoprotein require humoral and cell-mediated pathways to achieve tumor immunity (40.25)
Authors: Lowe Devin B , Shearer Michael H , Aldrich Joel , Jumper Cynthia A , Kennedy Ronald C
Published: January 2023

Malignant pleural mesothelioma (MPM) is an aggressive disease with survival prognosis less than one year. Interestingly, MPM samples have been reported to express the viral oncoprotein, Simian virus 40 (SV40) large tumor antigen (Tag). To this end, our laboratory has characterized protective SV40 Tag-specific immunologic mechanisms following prophylactic immunization in a murine experimental pulmonary metastasis model. Recent results indicate that ADCC functions as a primary in vivo anti-tumor mechanism since SV40 Tag antibody and Fcγ receptors are required immunologic-components in the observed protection from lung metastasis. We also find that with an antibody response due to vaccination, CD8+ T lymphocytes are required T cell subsets in the effector phase response of tumor challenge in vivo. The nature of the CD8+ T cell response appears to be CTL-driven based upon a systemic increase in Th1 cytokines by flow cytometry and tumor antigen-specific splenocyte production of IFN-γ when assessed by ELISpot and ELISA assays. In all, our results support the hypothesis that an SV40 Tag antibody-based ADCC pathway drives the activation of a cell-mediated response leading to protection from tumorigenic growth. These data also have implications for the construction of an effective MPM-specific vaccine that elicits broad-based immunity in individuals at high-risk for developing SV40 Tag-expressing tumor cells.
Cancer Immunoediting by the Innate Immune System (40.34)
Authors: Bui Jack D , Vermi William , Ngolab Jennifer , Arthur Cora , White J. Michael , Uppaluri Ravindra , Schreiber Robert D.
Published: January 2023

Although components of innate and adaptive immunity have been shown to work together to protect the host against cancer development and sculpt tumor immunogenicity (i.e., promote cancer immunoediting), it remains unclear whether innate immunity is capable of manifesting tumor-editing functions on its own. To address this question, we compared the immunogenicities of sarcoma cells derived from MCA-treated wild type (WT) mice, RAG2-/- mice lacking adaptive immunity, or RAG2-/- x IL-2Rγc-/- mice which lack natural killer (NK) cells and adaptive immunity. Consistent with our previous reports, all sarcoma cell lines from MCA-treated WT mice were poorly immunogenic and grew progressively when transplanted into syngeneic WT recipients. In addition, 40% of sarcoma cell lines from MCA-treated RAG2-/- mice displayed high immunogenicity and were rejected. Interestingly, MCA-sarcoma cells from RAG2-/- x IL-2Rγc-/- mice were highly likely to be immunogenic since 60% were rejected when transplanted into WT mice. Moreover, these highly immunogenic cell lines displayed delayed growth when transplanted into RAG2-/- mice and became heavily infiltrated with macrophages that expressed high levels of MHC class II and the glycolipid asialoGM1. This infiltration required IL-2Rγc function and resulted in tumor editing. Our results conclusively show that innate immunity can manifest IL-2Rγc-dependent tumor editing function in the absence of adaptive immunity. We propose that macrophages and NK cells collaborate as innate tumor editors.
Regulatory CD4+CD25+FOXP3+ T-cells can be Converted into Cytotoxic Effectors (40.36)
Authors: Peikert Tobias , Coughlin Kathleen , Van Keulen Virginia , Hansen Michael , Pease Larry
Published: January 2023

Background: Binding of the human IgM antibody B7-DC XAb to B7-DC on dendritic cells (DC) has profound immune modulatory effects. It enhances antigen presentation by DC, repolarizes T-helper (Th) 2 to Th1 cell responses, rapidly generates cytotoxic effectors and reprograms regulatory T-cells (Treg) into non-suppressive, cytokine producing autoimmune effectors. We hypothesized that co-culture of B7-DC XAb treated DC (DCXAb) with Treg results in the rapid generation of cytotoxic effectors.

Methods: Bone marrow derived DC from wild type C57BL6 mice (WT-B6) were treated for 24 hours with either B7-DC XAb or a control human IgM antibody in the presence of melanoma cell lysate (B16). Subsequently, DCXAb were co-cultured with Treg (CD4+CD25+FOXP3+), Non-Treg (CD4+, CD25-), cytotoxic CD8+ T-cells isolated from WT-B6 mice and Treg from CD8 knockout mice (CD8 -/-). After re-isolation these T-cells were used in cytotoxic T-lymphocyte (CTL) assays targeting B16 and Thymic lymphoma (EL4) tumor cells.

Results: Treg isolated from either WT-B6 mice or CD8 -/- mice co-cultured with B16 lysate treated DCXAb but not control antibody treated DC transformed into cytotoxic effectors targeting B16 but not EL4 tumor cells. Their level of cytotoxicity was comparable to that of CD8+ cells co-cultured with DCXAb. In contrast Non-Treg co-cultured with DCXAb did not kill in similar CTL assays.

Conclusion: Co-culture of Treg with antigen treated DCXAb rapidly generates antigen specific cytotoxic effectors.
Lentivector immunization stimulates potent CD8 T cell responses against melanoma self antigen tyrosinase related protein 1 and generates antitumor immunity in mice (41.10)
Authors: He Yukai , Liu Yanjun , Peng Yibing , Munn David
Published: January 2023

Recombinant lentivector immunization has been demonstrated to induce potent CD8 T cell responses in vivo. In this study, we investigated whether lentivector delivering a self/tumor Ag, tyrosinase related protein 1 (TRP1), could stimulate effective antitumor T cell responses. We found that immunization with lentivector expressing mutated TRP1 (muTRP1) Ag elicited potent CD8 T cell responses against multiple TRP1 epitopes. Importantly, the activated CD8 T cells effectively recognize wild type (wt) TRP1 epitopes. At peak times, as many as 10% of CD8 T cells were effector cells against TRP1 Ag. These cells killed wtTRP1 peptides-pulsed target cells in vivo and produced IFN-gamma after ex vivo stimulation. The CD8 T cell responses were long-lasting (3-4 weeks). Immunized mice were protected from B16 tumor cell challenge. In a therapeutic setting, lentivector immunization induced potent CD8 T cell responses in tumor bearing mice. The number of infiltrating T cells and the ratio of CD8:CD4 were dramatically increased in the tumors of immunized mice. The tumor infiltrating CD8 T cells (TIL) were functional and produced IFN-gamma. The potent CD8 T cell responses stimulated by lentivector immunization eliminated small 3 day subcutaneous B16 tumors and strongly inhibited the growth of more established 5 day tumors. These studies demonstrate that genetic immunization with lentivector expressing mutated self/tumor Ag can generate potent CD8 T cell immune responses and antitumor immunity that prevent and inhibit B16 tumor growth, suggesting that lentivector immunization has the potential for tumor immunotherapy and immune prevention.
Enhancing HER2/neu Specific T-cell Therapy by Targeting the Tumor Endothelium (41.11)
Authors: Lai Vy Phan , Buckanovich Ronald J , Gad Ekram , Bigdeli Laleh , Disis Mary L
Published: January 2023

Success of adoptive T cell therapy for cancer can be hindered by the tumor stroma, which consists of factors that not only promote tumor growth but also prevent egress of tumor-specific T cells into the tumor parenchyma. We recently demonstrated that T cell transit through tumor vasculature is regulated by the endothelin B receptor (ETBR). In addition, the endothelin axis plays a major role in tumor cell growth, apoptosis, angiogenesis and metastasis. The purpose of this study was to determine if, in breast cancer, targeting endothelin-1 via ETBR blockade might abrogate the endothelial barrier and increase T cell homing to tumors, thereby augmenting the efficacy of HER2/neu antigen specific Type 1 helper (Th1) cell therapy of spontaneous HER2/neu+ mammary tumors. By reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC), we found overexpression of ETBR in peritumoral stroma of spontaneous mammary tumors derived from HER2/neu transgenic mice. We treated tumor-bearing mice with HER2/neu specific T cells alone or HER2/neu specific T cells + BQ-788, a specific ETBR inhibitor peptide. The addition decreased tumor growth by 84%. Thus, in this model of HER2/neu mediated breast cancer, targeting the tumor endothelium can significantly enhance the clinical efficacy of HER2/neu specific Th1 cells. Studies are underway to evaluate the biological mechanisms induced by endothelin targeting that cooperate with T cell therapy in the resulting significant success of the combined treatment. The data provide a rationale for combining tumor antigen specific T cell therapy with endothelin blockade to induce an effective anti tumor response in breast cancer.
Generation of long-term protection from acute lymphoblastic leukemia (ALL) by an immune stimulant in a syngeneic murine model (41.21)
Authors: Seif Alix Eden , Barrett David M. , Brown Valerie I. , Grupp Stephan A. , Reid Gregor S.D.
Published: January 2023

ALL is the most common childhood cancer, and relapsed ALL remains a major cause of mortality. The success of stem cell transplant for relapsed ALL suggests immune therapy may be effective. We previously showed CpG ODN, which stimulate immune activity through TLR9, enhance allogeneic Th1 responses and reduce in vivo leukemic burden of primary human ALL xenografts. We are now evaluating anti-leukemia activity induced by CpG ODN in a pre-B ALL minimal residual disease (MRD) model. In mice given 106 syngeneic lymphoblasts, CpG ODN treatment universally induced remissions for >6 months; control mice died of leukemia at a median of 35 days (p=0.0001). CpG ODN treatment of Rag-1-deficient mice significantly decreased peripheral disease at day 28 (p<0.05) but did not induce long-term remissions (p<0.01). T cell-depletion after CpG ODN similarly abrogated protection from ALL, resulting in a median survival of 39 days (p<0.0001). Mice in remission after CpG ODN were protected from rechallenge with ALL blasts, while age-matched leukemia-naïve controls rapidly died of disease (p=0.0005). A protective effect is also noted in mice treated with CpG ODN versus PBS following standard ALL chemotherapy (p<0.03). To our knowledge, this is the first demonstration that CpG ODN induce a durable remission in an ALL MRD model by triggering an adaptive immune response, suggesting this treatment may have clinical utility in children with MRD.
Adoptive therapy of a fast growing tumor with reactivated memory T cells combined with anti-4-1BB eliminates tumors through effects of anti-4-1BB on transferred but not host cells (41.37)
Authors: Lin Gloria Hoi Ying , Liu Yuanqing , Ambagala Thanuja , Kwon Byoung S , Watts Tania H
Published: January 2023

The ability to expand memory T cells with cytokines ex vivo has greatly increased the practicality of adoptive immunotherapy for cancer. Central memory T cells generated in IL-15 have the advantage of longevity after subsequent in vivo transfer, whereas effector cells have the advantage of more immediate tumor cell killing. Here we report that in adoptive immunotherapy against a pre-established EG.7 tumor, a greater proportion of tumor bearing animals were cured with reactivated memory T cells as compared to animals that received central memory cells. Although central memory cells showed an initial survival advantage in the host, reactivated memory cells expanded more rapidly in the tumor, draining lymph node and spleen, resulting in increased accumulation over time. Co-administration of reactivated memory T cells with anti-4-1BB agonist antibody further potentiated the therapeutic effect. Anti-4-1BB therapy resulted in expansion of host NK, NKT, CD11c+ cells, CD4 and CD8 T cells as well as the adoptively transferred T cells. However, use of 4-1BB-deficient hosts showed that the expression of 4-1BB on adoptively transferred T cells was sufficient for the therapeutic effect. Thus, the combination of reactivated memory T cells and stimulatory anti-41BB antibody represents a superior immunotherapy for a rapidly growing cancer, largely through effects of anti-4-1BB on transferred effector T cells.
A novel form of 4-1BBL demonstrates better immunomodulatory activity than an agonistic anti-4-1BB Ab without Ab associated severe toxicity (41.4)
Authors: Schabowsky Rich-Henry , Elpek Kutlu G , Madireddi Shravan , Sharma Rajesh K , Yolcu Esma S , Bondura-Morgan Laura , Mittler Robert S , Shirwan Haval
Published: January 2023

Costimulatory molecules of the CD28 and TNFR superfamilies modulate innate, adaptive, and regulatory immune responses, and therefore agonistic ligands to these receptors have potential to serve as effective immunomodulatory components of therapeutic vaccines. However, the use of costimulatory agonists for vaccines in humans requires the generation of agonists that transduce appropriate stimulatory signals without toxic side effects. We hypothesized that signaling by natural ligands may have better efficacy and safety compared to agonistic Abs. Our laboratory recently developed a novel form of soluble 4-1BB ligand (SA-4-1BBL) which forms functional tetramers/oligomers. SA-4-1BBL demonstrated superior immunostimulatory activity than an agonistic 4-1BB Ab (3H3), and importantly lacked Ab associated toxicity as manifested by greatly enlarged spleen and LNs, non-specific T cell proliferation, hepatitis, and increased T cell and systemic inflammatory cytokine production. Ab associated toxicity did not involve Fc receptor or complement activation, and adoptive transfer studies demonstrate that CD8+ and CD4+ T cells were the direct targets of Ab mediated toxicity. Our findings support further development of this novel form of soluble 4-1BBL as an immunomodulatory component of therapeutic vaccines.NIH (R43 AI071618, R41 CA121665, R44 AI071618, and R43AI074176), KLCRP, and W.M. Keck Foundation.