bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-10 blockade resurrects T cell activity and eliminates a persistent viral infection (46.3)
Authors: Brooks David G , McGavern Dorian B , Oldstone Michael B
Published: January 2023

By definition persistent viral infections are accompanied by immune evasion, and most notably the failure to sustain robust T cell responses, responses that are essential to control of these infections. Identifying and understanding the immunoregulatory mechanisms that direct productive versus abortive T cell responses to viral infections is critical, but currently unclear. Using the model of persistent lymphocytic choriomeningitis virus (LCMV) infection, we found that upregulation of interleukin (IL)-10 early during infection induces T cell exhaustion and directly leads to viral persistence. Blockade of IL-10 activity induced sustained T cell function, the rapid elimination of an otherwise persistent virus infection and development of functional memory T cells capable of preventing re-infection. Antibody blockade of the IL-10 receptor during the chronic phase of infection restored T cell activity and eliminated an established persistent viral infection. Further, by manipulating this immunoregulatory molecule we significantly enhance immune responses to vaccination, indicating a potential “adjuvant” role to anti-IL10 immunotherapy. Thus, we propose a novel therapy to treat persistent viral infection by targeting host immunoregulatory molecules and reactivating antiviral T cells.
Murine AIDS requires CD154/CD40L expression by the CD4 T cells that mediate retrovirus-induced disease: Is CD4 T cell receptor ligation needed? (46.9)
Authors: Li Wen , Green William R
Published: January 2023

LP-BM5, a retroviral isolate, induces a disease featuring an acquired immunodeficiency syndrome termed murine AIDS (MAIDS). Many of the features of the LP-BM5-initiated disease are shared with HIV/AIDS. Our lab has shown that the interaction of B and CD4 T cells that is central to MAIDS pathogenesis requires ligation of CD40 on B cells by CD154 on CD4 T cells. Despite this strict requirement for CD154 expression, whether CD4 T cell receptor (TCR) occupancy, and the generation of TCR-associated “Signal 1”, are essential for the induction of MAIDS is unknown. To functionally block TCR engagement, transgenic (Tg) mouse strains with monoclonal TCR of irrelevant peptide/MHC specificities, all on MAIDS-susceptible genetic backgrounds, were tested: the study of a panel of TCR Tg CD4 T cells controlled for the possibility of serendipitous crossreactive recognition of virus-associated or induced-self peptide, or superantigen, MHC complexes by a given TCR. The data indicate that CD4 T-cell TCR ligation is not a necessity for these CD4 T cells to mediate MAIDS initiation and progression, and thus the induction of CD154 expression that is required of the “pathogenic” CD4 T cells must be occurring in the absence of TCR occupancy and classic generation on Signal 1.

This work was supported by National Institutes of Health Grants CA50157 and AI059580 (to W.R.G.).
DEC-205 antigen targeting at a dendritic cell-enriched site and CD40L-directed dendritic cell activation enhances CD4+ T cell priming. (47.12)
Authors: Mwangi Waithaka , Brown Wendy C. , Hope Jayne C. , Palmer Guy H.
Published: January 2023

To enhance DNA vaccine efficacy, we tested the hypothesis that inoculation of calves with a single dose of a DNA vaccine capable of DC recruitment, DC antigen-targeting and activation will prime significant CD4+ T cell responses. To test this hypothesis, we developed a DNA construct, pCC98MSP1, in which a DEC-205-specific single chain antibody was used for DC-targeting of an Anaplasma marginale MSP1a DR-restricted CD4+ T cell antigen. A DNA construct, pCD40L-ED, encoding CD40 ligand was used for DC activation and DNA-encoded Flt3L/GM-CSF were used for DC recruitment. Recombinant CC98MSP1 binds to DEC-205 in vitro and to skin DCs in situ. In a dose-escalation experiment, DR-matched calves were immunized with a single dose of pCC98MSP1 (0.25–3mg) mixed with pCD40L-ED, pFlt-3L and pGM-CSF (0.5mg each). Immunization with 0.25mg pCC98MSP1 primed CD4+ T cell responses detectable in fresh PBMC 1wk post-immunization (PI). A dose dependent CD4+ T cell response was detected and surprisingly, at 2wks PI, the response induced by 0.25mg was not statistically different from the responses induced by 0.5–3mg pCC98MSP1. The responses were maintained for >9wks PI and underwent rapid recall 4 days post-boost with 0.25mg pCC98MSP1. This data supports the hypothesis that immunization with a single dose of a DNA vaccine capable of DC recruitment, DC-antigen targeting and activation enhances vaccine efficacy.

Funding: USDA-CSREES 2005-01693
Novel vaccination (HVJ-liposome/HSP65 DNA+ IL-12 DNA against Tuberculosis) (47.15)
Authors: Okada Masaji , Kita Yoko , Nakajima Toshihiro , Kanamaru Noriko , Hashimoto Satomi , Nagasawa Tetsuji , Kaneda Yasufumi , Nishida Yasuko , Fukamizu Reiko , Tsunai Yoshie , Inoue Ruriko , Nakatani Hitoshi , Namie Yumi , Yamada Junko , Takao Kyoko , Asai Ritsuko , Asaki Ryoko , Tan Esterlina Babie , Sakatani Mitsunori
Published: January 2023

We have developed a novel tuberculosis (TB) vaccine; a combination of the DNA vaccines expressing mycobacterial heat shock protein 65 (HSP65) and interleukin 12 (IL-12) delivered by the hemagglutinating virus of Japan (HVJ)-liposome (HSP65+IL-12/HVJ). This vaccine provided remarkable protective efficacy in BALB/C mouse and guinea pig models compared to the BCG vaccine, on the basis of an induction of the CTL activity and improvement of the histopathological tuberculosis lesions, respetively. This vaccine strongly induced CTL against HSP antigen and M.tuberculosis antigens

Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis. This novel vaccine provided a higher level of the protective efficacy than BCG based upon the assessment of mortality, the ESR and immune responses. The combination of HSP65 + IL-12/HVJ and BCG by the priming-booster method showed a synergistic effect in the TB-infected cynomolgus monkey (100% survival). In contrast, the survival of the BCG Tokyo group was 33%. These data indicate that our novel DNA vaccine might be useful against M.tuberculosis for human clinical trials. [H-17-Shinko-5] of Research on Emerging and Re-emerging Infectious Diseases in Health Sciences Research grants from Japan.
Co-delivery of lymphopoietic cytokines in live Salmonella vaccines enhances the T cell response (47.17)
Authors: Feng Chiguang , Rodriquez Liliana , Davis Katherine , Pasetti Marcela
Published: January 2023

Live bacteria vaccine S. Typhi elicits strong mucosal and systemic immune responses. It is useful as a live vector to deliver heterologous antigens. Additionally, delivery of a eukaryotic expression vector by attenuated Salmonella leads to temporary expression of the coded gene in host cells after invasion. Because Salmonella invades preferentially macrophages and dendritic cells, the live bacteria could also be used to deliver immunomodulatory molecules to antigen-presenting cells. We hypothesize that co-delivery of lymphopoietic cytokine such as IL-2, IL-7 and IL-15, which has potential in promoting immune memory, enhances the efficacy of vaccination.

Murine IL-2, IL-7 and IL-15 were cloned into eukaryotic expression vector pCMS-EGFP, and introduced into S. Typhi(F1), which carries Y. pestis protective antigen F1. Mice were immunized intranasally with above derivative strains, and boosted with purified antigen F1 in alum adjuvant Alhydrogel.

This prime-boost strategy elicited strong mucosal and systemic antibody responses, as well as cell-mediated responses, e.g. antigen-specific proliferation and IFN-γ production of splenocytes. Inclusion of cytokine, however, further enhanced the cell-mediated responses. Antibody responses were not affected.

Our data strongly support our hypothesis that production of lymphopoietic cytokine during priming vaccination promotes memory T cell responses.
Immunogenicity of co-administered recombinant VSV vectors expressing GM-CSF and HIV-1 Gag in mice (47.25)
Authors: Wright Kevin James , Calderon Priscilla C , Guo Min , Natuk Robert J , Nasar Farooq , Johnson J Erik , Witko Susan , Clarke David K , Hendry R Michael , Parks Christopher , Eldridge John , Cooper David
Published: January 2023

Highly attenuated recombinant vesicular stomatitis virus (rVSV) vectors expressing HIV-1 Gag have been shown to be immunogenic in preclinical animal models. Here we explored the potential to enhance immunogenicity through co-delivery of either replication-competent (rVSV-N3CT9) or non-propagating (rVSV-Gstem) vectors expressing GM-CSF. A construct expressing both antigen and cytokine, Gstem-Gag1-GMCSF6, was also generated. BALB/c mice were injected i.m. with Indiana rVSV vectors and boosted 8 weeks later with New Jersey glycoprotein-exchange vectors. Co-delivery of 105pfu of N3CT9-GMCSF5 and 105 pfu of N4CT9-Gag1 resulted in Gag specific IFN-g responses that were approximately two-fold higher than responses elicited by 105pfu of N4CT9-Gag1 alone. These results were confirmed by CBA analysis of supernatants from peptide-restimulated cells. Interestingly, MHC-pentamer staining revealed no significant difference in the frequency of Gag specific CD8+ cells between the two immunization groups. Similar observations were made in tests of the Gstem vector, where 106pfu of Gstem-Gag1-GMCSF6 elicited a more robust IFN-g response after boost than 106pfu of Gstem-Gag1, while the percentage of Gag specific CD8+ cells remained equivalent. In conclusion, GM-CSF exerted immunomodulatory effects in vivo, distinguished here by the enhanced functional differentiation of vaccine elicited effector cells.

This work was supported by NIH contract NO1-AI-25458
Timing, dose and combination impact immune memory recall with TLR ligands (47.30)
Authors: Kumaraguru Uday , Woolard Stacie N , Nandakumar Subhadra
Published: January 2023

The hygiene hypothesis and the study of probiotic and prebiotic effects have emphasized the need to understand, and ultimately to manipulate, our physiological interactions with commensal flora, and with other transient but harmless organisms from the environment that affect immunoregulatory circuits. A long-term memory response is established following primary antigen exposure that stays more or less constant. It appears to adopt a set-point in magnitude but upon re-exposure the response is quicker and better and there is an upward shift in memory frequency. This new set-point is likely higher than the primary set-point and varies with individuals based on the exposure pattern to other microbes or its components.

Our investigations have been designed to test such differences of non-specific stimulation by PAMPs in lowering the threshold of activation. Accordingly, young mice were pre-exposed to TLR stimulation intermittently and later analyzed for its resilience to challenge with virus during later part of their life. Secondly, adult mice with pre-existing memory to virus were exposed to various TLR ligands in the presence and absence of cognate antigen and analyzed for their quality and frequency of the memory response.

Animals that are constantly reminded of the danger in a subtle way are better responders to a new agent exposure compared to the animals kept in sterile surroundings. However, immune memory recall with TLR ligands differed based on the dose, timing and combination of ligands used. Taken together, results provide insights into the role of hyper-sanitized environment versus PAMP mediated signaling in adaptive immune response and long term memory.
Active Hexose Correlated Compound (AHCC) Activates Immune Function to Decrease Bacteria Load in a Murine Model of Surgical Wound Infection (47.34)
Authors: Aviles Hernan O , O’Donnell Phyllis M , Orshal Julia M , Sonnenfeld Gerald
Published: January 2023

In spite of the use of modern techniques, infection continues to be a leading cause of death in patients subjected to severe trauma and surgical wounds. Previous studies have shown that AHCC, a natural compound, increases survival and prolongs the time to death in a murine model of surgical wound infection. This model simulates some of the conditions that occur after trauma or surgical procedures including systemic infection and food deprivation. To better understand the mechanisms involved in this model, food-deprived mice receiving either AHCC or the excipient were infected with sub-lethal doses of Klebsiella pneumoniae. Organ bacterial load, plasma levels of cytokines, and differential white blood cell counts were determined at different time points after infection. In contrast to control mice, which did not clear bacteria from organs, mice receiving AHCC had significantly reduced numbers of bacteria at day 5 and cleared bacteria entirely from the system at day 6. Levels of IL-12, TNF-α, and IL-6 peaked earlier in this group (day 3) compared with controls (day 5). AHCC also induced a significant increase of numbers of macrophages at day 2 and lymphocytes at day 3. Additional studies indicated that AHCC improved the function of peritoneal cells as measured by nitric oxide production and spleen cell proliferation affected by food deprivation in this model. Taken together, these results suggest that AHCC protects mice restoring the immune system and promoting effective clearance of bacteria and, consequently, a rapid recovery.

Funded in part by a grant from the Amino Up Chemical Company, Sapporo, Japan.
Blockade of PD-1/B7-H1 interaction restores antigen-specific CD8+ T cell responses in HCV core transgenic mice (47.8)
Authors: Lukens John R , Hahn Young S.
Published: January 2023

Impaired CD8+ T cells have been implicated to play a role in the establishment of HCV persistent infection. HCV has evolved multiple strategies to avoid T cell responses. HCV core protein has been shown to contain immunomodulatory functions. To determine HCV core-mediated regulation of antigen-specific CD8+ T cell responses in vivo, a transgenic mouse where the expression of core protein is directed to the liver using the albumin (Alb) promoter was generated. Using this HCV core transgenic murine model system, we demonstrated that core-expressing mice failed to clear adenovirus-LacZ (Ad-LacZ) infection in the liver. Particularly, the generation of Ad-LacZ specific CD8+ T cells was impaired in the liver of HCV core transgenic mice having suppression of the production of IFN-gamma and TNF-alpha by CD8+ T cells and alteration of the production of the cytolytic protease, granzyme-B. Interestingly, HCV core transgenic mice exhibited enhanced expression of the inhibitory receptor PD-1 by Ad-LacZ specific CD8+ T cells and its ligand B7-H1 following Ad-LacZ infection. Importantly, blockade of the PD-1/B7-H1 inhibitory pathway using a B7-H1 blocking antibody in core transgenic mice enhances effector function of CD8+ T cells and clearance of Ad-LacZ-infected cells. It suggests that the regulation of the PD-1/B7-H1 inhibitory pathway is crucial for HCV core-mediated impaired T cell responses and viral persistence/maintenance. Also, these findings implicate that manipulation of PD-1/B7-H1 pathway may be a potential immunotherapy to enhance effector T cell responses during persistent infection.
RTK-based vaccines elicit specific CD8+ T cell responses in HLA-A2 transgenic mice (48.1)
Authors: Komita Hideo , Mandic Maja , Kawabe Mayumi , Storkus Walter J
Published: January 2023

Many receptor tyrosine kinases (RTK) are overexpressed/dysregulated in advanced cancers, serving as negative prognostic indicators. We evaluated the immunogenicity of dendritic cell (DC)/RTK(EphA2, EGFR, Her2/neu)-based vaccines in HLA-A2 (HHD) Tg mice. To enhance the stimulatory capacity of BM-derived DCs, these cells were infected with a recombinant adenovirus encoding mIL-12p70 (Ad.IL12), prior to being pulsed with RTK-derived peptides. We also evaluated a cohort of DC co-infected with both Ad.IL12 and Ad.EphA2. Each of these DC preparations (vs. control DCs) were injected three times s.c. into HHD mice on a weekly schedule. DTH reactions were monitored 48–72h after each vaccination and splenocytes were harvested on d28 and d60 to assess specific CD8+ T cell reactivity (IFN-γ ELISA). Autoimmunity pathology in RTK+ normal tissues (i.e. kidney, liver, lung) were also evaluated for immune infiltration and tissue destruction by (immuno)histochemistry. Vaccines induced specific DTH responses and Tc1 responses in HHD mice, in association with an increased accumulation of CD8+ T cells around proximal endothelial cells (PEC) in the kidney and in the interstitial lung at d28, in the absence of tissue pathology. DC/RTK-based vaccines may promote specific Type-1 immunity that has the potential to eradicate RTK+ tumors, while sparing normal RTK+ tissues.

This work was supported by NIH R01 grant CA114071 (W.J.S.)
Regulation of Antigen Processing Molecules in Adenovirally Engineered Human Dendritic Cells (48.10)
Authors: Vujanovic Lazar , Whiteside Theresa L. , Ferrone Soldano , Butterfield Lisa H.
Published: January 2023

Multiple strategies utilizing the inherent immunostimulatory ability of dendritic cells (DC) are being tested as cancer vaccines. Our group has focused on recombinant adenoviral vectors (AdV) as vehicles for antigen engineering of DC in order to harness their immunostimulatory ability and activate multiple tumor antigen-specific T cell specificities. Previously we have found that AdV transduction can skew DC towards mature phenotype, and that cytokine-induced maturation of DC can upregulate the level of key components of antigen processing machinery (APM). In this study we have investigated the effects of AdV transduction on APM component expression levels in human DC and have compared these values to the level of APM components in immature (iDC) and in IFN-γ/LPS-matured (mDC) DC. The level of surface markers and of APM components in DC transduced with AdV was intermediate between that in iDC and in mDC, suggesting that AdV induces an intermediary level of DC maturation. Immunoproteasome subunits LMP2 and LMP7, peptide transporter TAP2 and chaperone tapasin are upregulated after AdV transduction. Interestingly, peptide transporter TAP1, which is upregulated in mDC, shows little change in AdV-infected DC when compared to iDC. Together, this data elucidates vital molecular changes in AdV-transduced DC that could influence T cell priming and that should be considered when designing effective cancer vaccines.

This work was supported by a Scientist Development Grant from the American Heart Association (#0330102N), the University of Pittsburgh Cancer Institute and the Henry L. Hillman Foundation (to L.H.B.)
Xenogeneic tyrosine hydroxylase DNA vaccination is effective against neuroblastoma (48.14)
Authors: Lode Holger N , Huebener Nicole
Published: January 2023

Neuroblastoma cells are derived from sympathetic neuroblasts and express high amounts of tyrosine hydroxylase (TH), the first step enzyme in catecholamine biosynthesis. We demonstrated that expression profiles of 68 neuroblastoma revealed stable TH gene expression independent of stage and MYCN amplification. Here, we report that a xenogeneic immunization with TH DNA vaccines induces an anti-neuroblastoma immune response in vivo.

For this purpose, three DNA vaccines were designed: one encoding for the whole coding sequence of the human TH gene (hTH) (cDNA). The second vaccine was constructed using the epitope prediction program syfpeithi: it comprises of three hTH derived epitopes with high predicted binding affinity to MHC class I with the epitopes separated by an AAY-spacer peptide. In order to amplify the immune response we designed a third DNA vaccine encoding for the hTH gene and single chain (sc)IL-12. The bifunctional characteristics of this construct, antigen expression and IL-12 bioactivity, were demonstrated in vitro.

The induction of protective immunity against neuroblastoma was determined in vivo using strain SL7207 of Salmonella typhimurium as an oral vehicle for the DNA vaccines. Interestingly, the hTH cDNA vaccine and the hTH minigene vaccine were equally efficient in inducing T-cell mediated suppression of subcutanous tumor growth and could effectively protect mice from spontaneous liver metastasis. Furthermore, the introduction of scIL-12 did not further amplify the anti-tumor immune response indicating the efficiency of the “danger signal” provided by the bacterial vaccine vehicle.

The results indicate that xenogeneic immunization is a promising concept for active immunotherapy in neuroblastoma patients.

Supported by DFG (Lo635/2).
Modulation of adenoviral vector immune responses through the over-expression of immune adaptor and viral immuno-modulatory genes (48.17)
Authors: Hartman Zachary , Yang Xiao Yi , Lei Gangjun , Amalfitano Andrea , Morse Michael , Lyerly Herbert Kim , Clay Tim
Published: January 2023

For the past two decades, adenoviral vector platforms have been used as both gene therapy vehicles and vaccine platforms in various human clinical trials. However, the use of these, as well as other viral vector platforms, has been tempered by the innate immune and adaptive immune responses elicited. Despite the development of advanced generation adenoviral vectors, the innate responses precipitated by these vectors have remained largely unchanged due to the particular nature of the viral capsid and infectious process. To more effectively use these vectors for different purposes, we constructed adenoviral vectors encoding overexpression cassettes for various TLR adaptors (MyD88 and TRIF), RIG-I pathway adaptor (MAVS), as well as immuno-modulatory viral genes (pp65) whose expression might accentuate or inhibit the innate immune response enabled by adenoviral infection. Our studies show that while all of these immuno-modulatory vectors were able to effectively enhance (TLR and MAVS vectors) or inhibit (pp65 vectors) the NF-kB and IFN-beta responses in vitro, only certain vectors were able to affect the adaptive responses elicited in vivo. While our findings show that vector manipulation can influence vector-specific innate and adaptive immune responses, they also offer evidence for the highly regulatory nature of the innate response, as evidenced by the limited impact of certain adaptor overexpression.
Immunization with PSA/anti-PSA complexes induces T and B cell responses to PSA in PSA-transgenic mice (48.19)
Authors: Schultes Birgit Corinna , Hou Fuxiang , Smith L. Mary , Nicodemus Christopher F. , Frelinger John F.
Published: January 2023

The murine MAb-AR47.47 (IgG1κ) recognizes all circulating forms of PSA and targets the epitope EPEEFLT. In vitro studies showed that human and murine dendritic cells process PSA more efficiently in immune complexes (IC) with MAb-AR47.47. IC induced CD4+ and CD8+ IFN-γ producing T cells, whereas PSA alone or PSA combined with a non-specific antibody mainly stimulated CD4+ T cells. We have further investigated the activation of PSA-specific immune responses with IC in male PSA-transgenic mice, which express human PSA in the prostate. Mice were immunized with PSA and MAb-AR47.47 alone or IC consisting of MAb-AR47.47 and PSA at various concentrations. To investigate the need for foreign antibody in inducing immune responses, rabbit and goat polyclonal anti-PSA antibodies or MAb-AR47.47-ovalbumin were tested in parallel. The mice only generated a weak T helper 2 response to immunization with PSA alone. Robust T helper 1 and 2, CTL and antibody responses were induced in mice immunized with AR47.47-PSA IC. Interestingly, IC with PSA and the polyclonal goat or rabbit antibodies or MAb-AR47.47-ovalbumin induced stronger T helper cell responses to PSA, but resulted in weaker CTL induction relative to IC with MAb-AR47.47. This data indicates that AR47.47-PSA IC can prime B cells, T helper cells and CTL against the tumor antigen PSA in vivo in a setting where PSA is a self antigen.
Sensitization of murine B16 melanoma cells with the HDAC inhibitor depsipeptide promotes immune cell-mediated tumor destruction (48.24)
Authors: Murakami Takashi , Sato Atsuko , Furukawa Yusuke , Kobayashi Eiji
Published: January 2023

Histone deacetylase inhibitor (HDACi) can restore gene expression that is aberrantly repressed in refractory cancer. Our recent DNA microarray analysis demonstrated that HDACi depsipeptide markedly augments expression of gp100/pmel17 melanoma antigen (15 fold), which is repressed in human melanoma cell lines. Herein, we set out to determine whether depsipeptide enhances tumor-specific T-cell-mediated killing of B16 murine melanoma cells. Firstly, by a quantitative assay of caspase-3/7 activity, we determined a sublethal dose of depsipeptide in vitro (ED50: ~5nM, after 24 hr), in which p21Waf1/Cip1, MHC class I and Fas were induced concomitantly with an increased level of histone H3 acetylation (~3 fold). Secondly, depsipeptide (5 nM) with Fas engagement synergistically promoted apoptosis in B16 cells (p<0.05, vs. depsipeptide; p<0.001, vs. FasL). Gp100-specific cytotoxic T cells also killed depsipeptide-treated B16 cells more efficiently than untreated B16 cells in vitro (p<0.01). Furthermore, in vivo metastatic growth of B16/F10 to the lung of C57BL/6 mice was synergistically suppressed by a combination of depsipeptide (2 mg/kg, i.p. only at day 1 following tumor implantation) with immune cell adoptive transfer (p=0.015, vs. PBS treatment, at day 20). Consequently, a transcriptional modulation strategy using HDACi might prove to be a useful adjunct in cancer immunotherapy.
Importance of Decay Accelerating Factor(CD55) in Tumor Avoidance of Immune Destruction (48.27)
Authors: Li Bing , Allendorf Daniel , Hansen Richard , Marroquin Jose , Cramer Daniel , Harris Claire , Yan Jun
Published: January 2023

Combined yeast derived β-glucan in conjunction with anti-tumor mAb therapy have demonstrated significant therapeutic efficacy. Therefore, we tested this strategy using human carcinomas implanted in immunocompromised SCID mice. Surprisingly, combined therapy achieved significant therapeutic efficacy in NCI-H23 human non-small cell lung carcinoma but failed in SKOV-3 human ovarian carcinoma. Immunohistochemistry analysis demonstrated markedly decreased levels of neutrophil infiltration and C5a deposition in SKOV-3 tumors compared to that in NCI-H23 tumors. Membrane complement regulatory proteins such as CD46 and CD55 were up-regulated in SKOV-3 cells. Blockade of CD46 and CD55 in vitro led to enhanced deposition of complement C3 activation product C3b on SKOV-3 cells. However, β-glucan-elicited in vitro CR3-dependent cellular cytotoxicity was only augmented in the presence of anti-CD55 mAb. To investigate the role of CD55 in the regulation of β-glucan mediated immunotherapy in vivo, anti-CD55 mAb was co-administered with yeast β-glucan and anti-Her-2/neu mAb to treat previously failed SKOV-3 tumors. Strikingly, significant tumor regression and long term survival were achieved in this model. Blockade of CD55 in vivo also restored neutrophil influx into tumor and C5a deposition. Thus, these studies identify a critical role for CD55 to regulate iC3b and C5a release and in turn to influence the recruitment of β-glucan primed neutrophils eliciting killing activity.
Two Chemotactic, Melanoma-Associated Proteins Stimulate Dendritic Cells (48.3)
Authors: Buzas Krisztina , Oppenheim Joost J , Howard OM Zack
Published: January 2023

IL-24 (melanoma differentiation associated gene 7 product) was initially identified as a melanoma associated antigen. IL-24 is actually a rapidly acting Th1 cytokine, IL-10 family member, induces selective apoptosis of tumor cells in vitro, and its expression correlates with melanoma tumor progression. The melanoma-associated antigen, gp100, mediates intracellular trafficking steps that regulate melanosome biogenesis. We have previously observed that self-antigens; including autoimmune and tumor antigens, induce cell migration by interacting with chemokine receptors. Further, we have reported that recombinant gp100 induces cell migration by interacting with CCR2. To further identify domains likely to be responsible for gp100 induced migration, 20 amino acid peptides spanning the first 140 amino acids of the mature human gp100 were synthetized and tested. One of the peptides, P3859, induces cell migration. We tested the chemotactic activity of rmuIL-24 for CCR2 positive monocytes. We found that rmuIL-24 induced human monocyte migration at 0.125–0.0125 ng/ml. Pertussis toxin, a Gi coupled protein receptor inhibitor, can reduce this effect, while a JAK inhibitor blocks the cell migration toward IL-24, suggesting a role for IL20R2 in the chemotactic signal transmitted by rmIL24. We tested the effect of the gp 100 melanoma antigen on IL-24 production by bone marrow derived dendritic cells. Data showed that gp100, P3859, and supernatant from B16 cells induced IL-24 transcription and expression by DC. These studies indicate that gp100 and rmuIL24 share a chemotactic activity and suggest that a crosstalk between CCR2 and IL20R2.
CD8 T cells with high avidity TCR-ab specific for serine protease self-determinants for use in adoptive immunotherapy of human leukemia (48.35)
Authors: Rusakiewicz Sylvie , Kant Shreya , Lizee Gregory , Rabinovich Brian , Wieder Eric , Lu Sijie , Hwu Patrick , Molldrem Jeffrey J.
Published: January 2023

Specific cytotoxic T-lymphocyte (CTL) immunity against the serine protease-derived PR1 peptide has been correlated with clinical response (CR) to interferon-α, allogeneic bone marrow transplantation or PR1-peptide vaccination in leukemia patients. However, some patients failed to demonstrate a CR, despite an immunological response. Patients with CR have functional PR1-CTL with a preferentially higher avidity T cell receptor (HTCR). These HTCR PR1-CTL were more susceptible to apoptosis by high PR1 peptide expression on leukemia cells compared to low avidity (LTCR) PR1-CTL, suggesting deletional tolerance mediated by the leukemia cells. We hypothesized that the adoptive transfer of HTCR PR1-CTL could induce remission in leukemia patients. Clonal, HTCR and LTCR PR1-CTL were elicited from an HLA-A2 healthy donor using low (2 μM) or high (200 μM) concentration of PR1 peptide, respectively. TCR affinity of these clones correlated with tetramer fluorescence intensity and importantly, effector function. Jurkat and primary T cells transduced with HTCR or LTCR by lentivirus vectors expressed cell surface PR1-specific TCR complex and specifically recognized the HLA-A2/PR1 tetramers. HTCR-αβ genes transduction represents a powerful strategy for adoptive immunotherapy in leukemia patients.
Alpha-enolase elicits specific pancreatic adenocarcinoma specific-T cell responses in healthy donors and in tumor patients (48.4)
Authors: Novelli Francesco , Tomaino Barbara , Ciccarelli Marianna , Capello Michela , Giovarelli Mirella , Novarino Anna , Cappello Paola
Published: January 2023

We have demonstrated that sera from pancreatic ductal adenocarcinoma (PDAC) patients contain IgG autoantibodies to α-enolase, a glycolytic enzyme that also works as surface plasminogen receptor. As this implies that α-enolase elicits a B-cell dependent humoral response in vivo in PDAC patients, its ability to induce a T cell mediated response to PDAC was investigated. Here we show that α-enolase-pulsed dendritic cells were capable to specifically stimulate healthy autologous T cells to proliferate, secrete IFN-γ and lyse PDAC cells. In vivo, α-enolase-specific T cells completely inhibited the growth of PDAC cells in immunodeficient mice. In PDAC patients, and in particular in those with circulating IgG autoantibodies to α-enolase, the existence of memory T cells to α-enolase was also demonstrated. As a whole, these results indicate that α-enolase elicits a PDAC-specific humoral and cellular responses. The sum of its features makes α-enolase a promising candidate for new immunotherapeutic strategies in the cure of PDAC that lacks until now of efficacious therapies to associate the conventional ones.
A novel role of myeloid related protein in dendritic cell differentiation (49.11)
Authors: Cheng Pingyan , Yu Bin , Nadaf Sorena , Carbone David P. , Nacken Wolfgang , Sorg Clemens , Gabrilovich Dmitry I.
Published: January 2023

Abnormal differentiation of dendritic cell (DC) is a critical factor contributing into ineffective immune response in cancer. We have searched for the genes that could be involved into tumor-induced defects in DC differentiation and found that tumor-derived factors consistently up-regulated expression of myeloid related protein 14 (MRP14) and its partner MRP8 in hematopoietic progenitor and myeloid cells during DC differentiation. Normal DC differentiation was associated with dramatic down-regulation of MRP14/8, whereas accumulation of immature myeloid cells was associated with persistently high levels of these proteins. Overexpression of these proteins in embryonic stem cells resulted in inhibition of DC differentiation and accumulation of immature myeloid cells able to form myeloid colonies. It is known that Gr-1+ myeloid-derived suppressor cells (MDSC) frequently accumulate in tumor-bearing mice and during in vitro DC differentiation in the presence of tumor-derived factors. We have found that in tumor-bearing mice MRP14/8 were exclusively accumulated in MDSC. Accumulation of these proteins was controlled by up-regulation of STAT3. MRP14/8 knockout mice had normal level of DCs or MDSC suggesting that MRP proteins were not required for accumulation of MDSC in cancer. Thus, this study demonstrates that tumor-induced up-regulation of MRPs inhibits DC differentiation, which may suggest a novel molecular mechanism of tumor-induced abnormalities in myeloid cells in cancer.
Understanding the Interplay of the Human Immune System with Cancer in Humanized Mice (49.17)
Authors: Pedroza-Gonzalez Alexander , Aspord Caroline , Gallegos Michael , Tindle Sasha , Burton Elizabeth , Marches Florentina , Banchereau Jacques , Palucka Anna K.
Published: January 2023

The paucity of in vivo models of human disease represents a major drawback in translational research. Indeed, the results obtained in mice cannot always be directly extrapolated to humans because of differences between the species. We have used humanized mice to study the human immune system, particularly DCs in vivo in human cancer. There, nonobese diabetic/LtSz-scid/scid β2m−/− mice engrafted with human CD34+ hematopoietic progenitors develop all subset of human DCs. They also develop B cells but T cells need to be adoptively transferred. In this model, we implanted human tumor cell lines derived from melanoma and breast cancer. Breast cancer attracts human dendritic cells and polarizes them to prime naïve CD4+T cells to secrete IL-13. CD4+T cells accelerate tumor development, which can be inhibited with IL-13 antagonists. CD4+T cells secreting IL-13 can be identified in tumor samples from breast cancer patients. Frozen tissue sections reveal IL-13 staining on breast cancer cells. The expression of phosphorylated STAT6 suggests that actually IL-13 delivers signals to breast cancer cells. Thus, breast cancer promotes skewed DC maturation leading to polarized adaptive immune response that facilitates tumor development. This model can thus be used to decipher pro-cancer mechanisms thereby guiding us in the development of novel anti-cancer therapies.

Supported by U19 AIO57234, RO-1 CA89440, CA78846 and CA85540
Increased Levels of Circulating CD4+CD25+ T cells (T-regs) Correlate with Clinical Cancer Stage And Inversely Correlate with CD8+ T cell Frequency (49.18)
Authors: Al-Khami Amir , Montero Alberto J. , Salem Mohamed L. , Vissage Kristi , Cole David J. , Diaz-Montero C. Marcela
Published: January 2023

Regulatory T cells inhibit immune responses mediated by T cells. Increased circulating levels of T-regs in patients (pts) have been reported in some solid tumors. Moreover, increased apoptosis among circulating CD8+ T cells has been reported in cancer pts. The goal of our study was therefore to determine how levels of circulating T-regs and CD8+T cells correlate with clinical cancer stage in a diverse cohort of pts. Flow cytometry analyses were performed on whole blood from 14 normal healthy controls and 35 cancer pts with newly diagnosed tumors prior to any therapy (stages: I/II, n=12; III/IV n=23). Although no significant difference was observed in T-regs between normal and stage I/II cancer pts (4.36% vs. 4.92%), levels were significantly increased in stage III/IV pts (7.26%; p=0.039). Moreover, there was an inverse correlation between CD8+T cell frequencies and clinical cancer stage, with stage III/IV pts having the lowest levels relative to normal (3.97% vs. 7.01%; p=0.036). In conclusion, T-regs were increased only in pts with advanced stage III/IV cancer and percentage of total CD8+T cells inversely correlated with clinical tumor stage. Thus, increased levels of T-regs and decreased levels of CD8+ T cells might contribute to cancer-mediated immunosupression.
Osteopontin induced STAT1 ubiquitination and degradation enhances cell proliferation (49.2)
Authors: Gao Chengjiang , Guo Hongtao , Kuo Paul C.
Published: January 2023

Signal transducer and activator of transcription 1 (STAT1) is a very important downstream component in interferon signaling required for the maximum induction of IFN responsive genes. IFN/STAT1 signaling composes a very important tumor-suppressor system to protect hosts against tumor development. At the same time, tumors can become insensitive to IFN/STAT1 signaling and thereby escape immune detection and elimination. But, the genetic mechanisms employed by tumors to become IFN/STAT1 insensitive was poorly defined. Osteopontin (OPN) is a pleiotropic cytokine that participates in a wide range of biological processes, including bone remodeling, inflammation and cancer metastasis. We found OPN can induce STAT1 ubiquitination and degradation in breast cancer cells. In addition, we identify STAT- interacting LIM (SLIM) protein as the STAT ubiquitin E3 ligase critical for STAT1 degradation in this setting. Consequently, p21WAF1/CIP1 expression was down-regulated. Therefore, cancer cells showed very strong proliferation ability upon IFN-gamma stimulation. Given that STAT1 is a tumor suppressor and OPN is highly expressed in metastatic cancers, these findings maybe can shed light about how invasive cancers can escape from tumor surveillance.

This work was supported by National Institutes of Health Grants AI44629, GM65113, and GM069331.
Recognition and cleavage of cryptic recombination signal sequences identified from lymphoid malignancies (49.20)
Authors: Zhang Ming , Swanson Patrick C.
Published: January 2023

Immune repertoire diversity is largely achieved by V (D) J recombination, during which antigen receptor genes are assembled from arrays of component gene segments by site-specific DNA rearrangement. This process is initiated when the RAG proteins introduce DNA double-strand breaks (DSBs) at recombination signal sequences (RSSs) abutting antigen receptor coding segments. However, the RAG proteins occasionally mistarget DNA sequences outside of antigen receptor loci and promote chromosomal translocations and deletions, which are among the major events that initiate neoplasia in lymphoid organs. Previous studies have identified various RSS-like sequences involved in chromosomal translocations, including LMO2, TAL1, Ttg-1, and Hox11, and the interstitial deletion at 1p32 involving SIL/SCL. Here we have used electrophoretic mobility shift assays and in vitro cleavage assays to study RAG-mediated binding and cleavage to the oligonucleotide substrates containing these cryptic RSSs. We find that RAG proteins can cleave LMO2, TAL1, Ttg-1, and SIL in vitro, and introduce DSBs at these sites which can be detected by ligation-mediated PCR in cell culture. In contrast, Hox11 and SCL are nicked but fail to support DSB formation both in vitro and in cell culture. These data raise the possibility that Hox11 and SCL may be cleaved by an as-yet unidentified mechanism.

This research is supported by American Cancer Society grant RSG-01-020-05-LIB to P.C.S.
The role of immature myeloid suppressor cells (IMSCs) in transgenic mammary tumor chemoprevention and therapy by Celecoxib (49.21)
Authors: Talmadge James E , Donkor Moses , Scholar Eric , Singh Rakesh , Dafferner Alicia , Hoke Traci , Westphal Sherry , Solheim Joyce
Published: January 2023

Immune manipulation can control tumor initiation and growth while tumor growth may suppress host immunity. In our studies of tumor–host interactions, we observed a tumor associated expansion of IMSCs and suppression of T cell number and tumor infiltration. Further, the cyclooxygenase-2 (COX-2) inhibitor, Celecoxib had chemopreventative and as shown for the first time, therapeutic activity for mammary tumors in HER-2/neu transgenic BALB/c mice. Tumor growth was associated with a significant decrease in T cells and a significant increase in IMSCs (CD11b+Gr1+). In addition type 1 cytokine transcripts were depressed, while transcription levels of COX-2, arginase-1 (ARG1), nitric oxide synthetase-2 (NOS2), granulocyte (G) and G-monocyte colony stimulating factor, and vascular endothelial growth factor-alpha (VEGF-a) were increased in the preneoplastic lesions, tumors, and spleens and to a greater extent tumor infiltrating leukocytes. Celecoxib chemoprevention reduced the changes in IMSC and T cell numbers and cytokine and enzyme transcription levels. We posit that the increase in IMSCs may be due to the increased levels of COX-2, VEGF-a and growth factor(s), while the loss of T cell numbers and function maybe due to the heightened levels of ARG1 and NOS2. Further, as Celecoxib administration does not increase T cell infiltration of tumors or decrease IMSC infiltration, the changes in cytokine and growth factor levels maybe critical to the improved clinical outcomes. Together, these studies suggest an important role for IMSCs and its regulation by Celecoxib in cancer induction, and therapeutic intervention.

Supported in part by funding from NIH grant 5 RO1 AT001739, Eppley Cancer Center and Nebraska DHHS LB506.
CCR9-CCL25 interaction mediates breast cancer cell survival via Akt activation (49.22)
Authors: Johnson-Holiday Crystal , Singh Shailesh , Johnson Erica , Singh Udai , Lillard James W.
Published: January 2023

Breast cancer (BrCa) is one of the most frequently diagnosed cancers and the second leading cause of cancer-related deaths in the U.S. Most cancer-related deaths are due to metastasis. In addition, BrCa cells readily become resistant to a wide variety of chemotherapeutic agents, which is a major obstacle in the treatment of BrCa. Tumor cell migration and metastasis share similarities with leukocyte trafficking, which is regulated, in part, by chemokines and their receptors. We have previously shown that CCR9 is functionally expressed by BrCa cells and its expression is enhanced by the presence of its ligand, CCL25. This study characterizes the role of CCL25-CCR9 cell-signaling cascades involved in BrCa cell resistance to cisplatin. This drug is often ineffective due to cancer cell resistance and the development of anti-apoptotic mechanisms. In this regard, CCR9-CCL25 interaction plays a role in immature T-cell survival through PI3K and G ai protein-dependent activation of Akt/protein kinase B. Activated Akt phosphorylates multiple downstream targets that are involved in cell survival signaling, which include glycogen synthase kinase (GSK-3), forkhead transcriptional factor (FKHR), and caspase-9. We examined the effect of CCR9-CCL25 interactions on BrCa cell lines MDA-MB-231 and MDA-435, with or without cisplatin treatment. CCR9-CCL25 interactions decreased cisplatin-induced apoptosis. These interactions also modulated the activation of cell signaling cascades involved in cell survival. These studies provide important new information regarding the cellular and molecular mechanisms that mediate BrCa cell survival.
Synergistic effect of VR1 agonist Capsaicin and its antagonist IRTX, in tumor cell apoptosis: implication for involvement of VR1 (49.24)
Authors: Ghosh Amiya Kumar , Basu Sreyashi
Published: January 2023

Capsaicin (CP) is the principal pungent ingredient of red peppers. The CP receptor, VR1 has been shown to be highly expressed by nociceptive neurons in dorsal root and trigeminal ganglia. Recently we have demonstrated that intratumoral administration of CP into a preexisting tumor results in retarded progression of the injected tumor. Here we report that tumor cells express VR1 and treatment with CP leads to tumor cell death by apoptosis as evidenced by positive TUNEL staining. Unlike tumor cells normal embryonic fibroblasts (MEFS) do not succumb to apoptosis by this treatment. CP treatment induced up-regulation of reactive oxygen species (ROS) and Caspase-3 activation in a dose-dependent manner. Further, we observed that VR1 antagonist I-RTX has synergistic apoptotic effect with CP like a partial agonist of VR1, indicating the involvement of VR1 in this apoptotic event. We also report that CP treatment leads to degradation of FAF1 (Fas associated factor1) a VR1 associated protein in a dose-dependent manner. In summary, our data suggests that VR1 engagement and subsequent degradation of FAF1 is one of the mechanisms of CP mediated apoptosis in tumor cells.
Failure of vSAg29-responsive CD4+CD25+ Treg cells to inhibit CD4+CD25− T cells resides in their differential regulation of cytokine, chemokine and toll-like receptor family genes (50.1)
Authors: Li Yu , Tsiagbe Vincent K
Published: January 2023

Endogenous mammary tumor virus (Mtv29)-encoded superantigen (vSAg29) mediates “reverse immune surveillance” that characterizes SJL lymphoma (RCS) development. CD4+Vβ16+ vSAg29-responsive T cells provide growth factors (notably IL-5 and IL-4) for RCS growth. The vSAg29-responsive CD4+ T cells belong to both CD4+CD25+ (Treg) and CD4+CD25− (non-Treg) populations. Unlike the known inhibitory ability of Treg cell on non-Treg cell responses to nominal antigens, Treg cells responding to vSAg29 fail to inhibit response of vSAg29-responsive non-Treg cells. This unique behavior of vSAg29-responsive T cells was found to be linked to their differential expression of cytokine, chemokine and toll-like receptor family genes. Treg cells responding to vSAg29 significantly inhibited the production of IL-4, IL-5, IL-10, IFN-γ, CCR4, CCL17, TOLLIP, UBE2N, NFKBIB, by CD4+CD25− cells. On the other hand, the addition of CD4+ Treg cells to non-Treg cells responding to vSAg29, resulted in enhanced production of mRNA for MIF, IL-10R, BCL6, CXCL4 (PF4), MAPK8 (JNK1), UBE2V1.

Research funded by CA10273 from NCI.
Hsp70-containing exosomes - potent stimulators of the innate immune system (50.19)
Authors: Multhoff Gabriele
Published: January 2023

Depending on their intra- and/or extracellular localization heat shock proteins (HSP) either mediate protection against stress-induced cell damage or act as danger signals stimulating the adaptive and innate immune system (1–3). However, it remained elusive how cytosolic HSPs become externalized and thus communicate with immunocompetent effector cells. Several groups demonstrated that a variety of different cell types, including tumor cells, have the capacity for an active release of Hsp70 in detergent-soluble vesicles (4,5). Biophysical properties including floating properties (1.17g/ml) correlating with a maximum acetylcholine esterase activity characterized them as exosomes (3–5). Profiling of luminal proteins revealed that tumor-derived exosomes contain cytosolic proteins but lack ER-residing proteins. An exosomal enrichment of the small GTPase Rab-4 documented their intracellular transport route from the early endosomal compartment to the plasma membrane. Exosomes originating from Hsp70 plasma membrane-positive tumors present Hsp70 on their exosomal membrane. In line with these findings only Hsp70 surface-positive exosomes but not their negative counterparts had the capacity to stimulate the innate immune system (3). These data provide an explanation how the innate immune system might become activated by tumor-derived exosomal Hsp70 in vivo.