bluuebunny/crossref_metadata_2025 huggingface dataset:
Memory T cells Require Costimulation and Dendritic Cell Migration to Purge an Established Persistent Viral Infection (47.7)
Authors: McGavern Dorian B. , Lauterbach Henning , Truong Phi
Published: January 2023

Adoptive immunotherapy relies on transfer of exogenously derived pathogen-specific (memory) T cells and is a highly successful approach to purge an established persistent viral infection. For example, administration of adoptive immunotherapy to mice persistently infected with lymphocytic choriomeningitis virus (LCMV) (carrier mice) results in systemic eradication of the virus, including noncytopathic clearance of persistently infected CNS neurons. By visualizing GFP-tagged LCMV-specific memory CD8+ T cells on brain reconstructions, it was revealed that cytotoxic lymphocytes (CTL) distributed throughout the CNS within 8 days after immunotherapy. Flow cytometric and microscopic analyses revealed that conventional dendritic cells arrived in the CNS concurrent with the memory CTL and induced anti-viral cytokine production (e.g., TNF-alpha). When adoptive immunotherapy was performed in carrier mice depleted of DCs or with memory T cells deficient in TNF-alpha, the adoptively administered memory T cells failed to purge the persistent infection. We further demonstrated by employing CTLA-4-Fc blockade and CCR2 deficient recipients that memory CTL rely on costimulation and DC migration, respectively, for optimal secondary expansion and efficient viral clearance. These studies underscore the importance of DC mobility and memory CTL-DC interactions in the clearance of a persistent viral infection.
Codelivery of GM-CSF by hydrogel overcomes non-responsiveness to hepatitis B vaccine through recruitment and activation of dendritic cells (47.9)
Authors: Tao Mi-Hua , Chou Ho-Yuan , Wu Pin-Yi
Published: January 2023

Nonresponsiveness to hepatitis B vaccine, composed mainly the major hepatitis B surface antigen (HBsAg), has been described in approximately 5–10% of healthy immunocompetent subjects. The underlying cellular and molecular events for the nonresponsiveness are not completely understood, but are likely associated with impaired T helper cell responses to HBsAg in subjects with particular HLA-DR alleles. In this study, we describe a novel formulation of HBsAg vaccine containing GM-CSF in a thermosensitive biodegradable copolymer (hydrogel) system, acting as a sustained-release matrix for the loaded proteins. In responder mouse strains, hydrogel HBsAg/GM-CSF vaccine elicited high titers of anti-HBs antibody and strong T-cell proliferative responses. Importantly, hydrogel HBsAg/GM-CSF vaccine also elicited strong anti-HBs antibody and T-cell immune responses in B10.M mice, a mouse strain with H-2 gene-linked nonresponsive phenotypes. The local and sustained release of GM-CSF is required for its immunostimulatory function in nonresponder mice, because administration of hydrogel/HBsAg and hydrogel/GM-CSF in separate locations or a free mixture of HBsAg and GM-CSF failed to induce detectable humoral and cellular immune responses. Analysis of draining lymph nodes of hydrogel HBsAg/GM-CSF-treated mice revealed an elevated number of CD11c+ dendritic cells with enhanced expression of CD80 and CD86. These results suggest that the local adjuvant activity of GM-CSF helps nonresponder mice generate T helper cells, which subsequently provide help for B cells to produce anti-HBs antibodies, by promoting DC migration to and activation in the draining lymph node. This approach may be applicable to design of vaccines for a variety of other diseases.
Immunomodulator AS101 potentiates apoptosis on Ha-Ras transformed cells which correlates with suppression of a survival protein Akt (48.20)
Authors: Sredni Benjamin , Eliyahu Siona , Lander Harry , Albeck Michael , Segal Shraga
Published: January 2023

The Ras family is the most widely mutated group of human proto-oncogene. Previously, we found an organic Tellurium compound, AS101, which exhibits immunomodulatory activity and strong anti-tumor effects. Whether these properties can be exploited for cancer therapy was examined using v-Ha-Ras transformed and V-mos transformed fibroblasts cells. In this study AS101 appeared to selectively block ~90 % of the proliferative growth of v-Ha-Ras transformed cells, while not affecting the growth of V-mos transformed cells. Furthermore, the proliferative growth inhibiting effect of AS101 was prevented when the activity of p21Ras was blocked by expression of a dominant negative mutant 116y of the v-Ha-Ras or by treating Ha-Ras transformed cells with a farnesyltransferase inhibitor. The anti-proliferative effect of AS101 was accompanied by morphological changes typical of apoptosis i.e.: membrane blebbing, condensation of nuclear chromatin, and formation of apoptotic bodies. AS101-induced apoptosis of v-Ha-Ras transformed cells correlated with suppression of Akt protein expression, activation of caspases, and induction of the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1. Thus, our results suggest that AS101 can be used to effectively treat tumors which, are induced due to an oncogenic mutation in p21Ras encoding gene and to selectively block oncogenic function of mutated Ha-Ras in human tumors.
CpG DNA augments dendritic cell-based therapy of colon cancer in transgenic mice (48.21)
Authors: Saha Asim , Chatterjee Sunil K. , Foon Kenneth A. , Celis Esteban , Bhattacharya-Chatterjee Malaya
Published: January 2023

Induction of potent and sustained anti-tumor immunity depends on the efficient activation of CD8+ and CD4+ T cells. Immunization using dendritic cells (DC) loaded with tumor Ag has shown promising results in cancer immunotherapy. Recently we have reported that in DC based immunization protocol in therapeutic setting with 7-day-old established MC-38-CEA-A2Kb tumors, mice (transgenic for both CEA and HLA-A2) immunized with agonist peptide for CEA691 (YMIGMLVGV, peptide-1) or mice immunized with agonist peptide for CAP-1 (YLSGADLNL, peptide-2) along with 3H1 (anti-idiotype Ab mimicking CEA) induced significant increase in survival with complete tumor eradication in 50% and 67.5% of the mice respectively. In this study, we attempted to augment anti-tumor immunity by the combination of 3H1-pulsed DC plus peptide-1-pulsed DC or peptide-2-pulsed DC along with immuno-stimulatory CpG (ODN–1826) in a therapeutic setting. Co-administration of CpG ODN provided significant increase in tumor free survival compared with mice immunized with peptide-pulsed DC plus 3H1-pulsed DC alone. Combined vaccination with CpG ODN resulted in increased secretion of Th1 cytokines such as IFN-γ and TNF-α, and HLA-A2-restricted CEA-specific CTL responses were also enhanced. Thus, the combination of Ag-loaded DC and CpG ODN is an effective vaccination strategy for future clinical application.

Supported by the NIH grant RO1 CA104804.
HLA Class II Protein Expression in Prostate Cancer Cells (48.22)
Authors: Haque Azizul , Younger Austin R , Amria Shereen , Geffrey Whitney A , Goldstein Oliver G , Norris James S
Published: January 2023

Prostate cancer is the second most commonly diagnosed cancer in men. Presently, there is no effective treatment for patients who develop recurrence, or for those who have metastatic disease at the time of diagnosis. Recent evidence suggests that reduced expressions of target protein antigens as well as HLA molecules are the predominant immune escape mechanisms of malignant prostate tumors. The purpose of this study was to investigate the prospect of antigen specific immunotherapy against prostate cancer via the HLA class II pathway of immune recognition. Here, we show for the first time that prostate cancer cells express functional HLA class II proteins that are recognized by T cells. We also show that prostate cancer cells can be induced with IFN-γ to express high levels of class II proteins as well as an enzyme, lysosomal thiol reductase (GILT), that may facilitate enhanced class II antigen presentation and CD4+ T cell recognition. Select prostate tumors can be transduced with HLA class II molecules resulting in efficiently presented antigenic peptides that also stimulate CD4+ T cells. These data suggest that prostate cancer cells can be targeted via the HLA class II pathway, and that genetically manipulated prostate tumor cells can be employed for direct antigen presentation, and CD4+ T cell mediated tumor therapy.
IDENTIFICATION OF AN ANTIGENIC AND IMMUNOGENIC MOTIF EXPRESSED BY TWO 7-MER RITUXIMAB-SPECIFIC CYCLIC PEPTIDES. (48.26)
Authors: Perosa Federico , Favoino Elvira , Merchionne Francesca , Dammacco Franco
Published: January 2023

Two 7-mer cyclic peptides, bearing the antigenic motif recognized by the anti-CD20 mAb Rituximab and differing because of motif-surrounding amino acids, inhibit the binding of Rituximab to raft-associated CD20 and Rituximab-induced membrane ceramide on human lymphoid DAUDI cells. These peptides displayed different immunogenic profiles, in that antibodies recognizing CD20 were induced in two and five out of five BALB/c mice immunized with Rp-15-C and Rp13-C respectively. Analysis of immunogenic motif, performed by panning a 7-mer phage display peptide library with purified anti-peptide IgGs, showed that the motif defined by anti-Rp15-C mostly included amino acids surrounding the Rituximab-specific antigenic motif “ANPS”, whereas that defined by anti-Rp13-C was “NPS”. These data showed that the motif-surrounding amino acids can markedly influence the specificity of antibodies, even when elicited with a short 7-mer peptide. Since anti-peptides antibodies analyzed are IgG, their specificity is likely to reflect how peptides are processed at the T cell level and suggests that, within a short peptide, the motif defined by T cells during the recognition phase may be different from that recognized by these cells upon their stimulation. Our findings can explain the failure of most peptide-based immunotherapy in cancer and autoimmune diseases and suggest strategies to implement the specificity of peptides-induced antibodies against the target antigen
Engineering T cell receptors for potential therapeutic applications (48.28)
Authors: Richman S A , Aggen D H , Stone J D , Weber K S , Dossett M L , Donermeyer D L , Mariuzza R A , Allen P M , Greenberg P D , Kranz D M
Published: January 2023

Using T cell receptors (TCRs) for potential therapeutic applications provides an opportunity to target an expanded array of antigens, compared to those now targeted with monoclonal antibodies. Relative to antibodies, TCRs typically exhibit reduced solubility and lower affinity, properties that hamper their production and efficacy. To improve these parameters, we have developed strategies using yeast display engineering. Several different TCRs were cloned as single chain (sc) TCRs and VαCα/VβCβ heterodimers, as each of these display scaffolds has unique advantages for engineering and potential applications. Display of scTCRs, unlike that of scFv fragments, required mutagenesis and selection. Analysis of a collection of mutants revealed distinct regions of the scTCR that require mutations to allow yeast surface display, as well as solubility of purified protein. Although the sc format has several potential advantages such as increased tissue penetration, mutagenesis and selection of displayed mutants can present a technical challenge. To overcome this obstacle, we have recently explored the role of the C domains in driving heterodimer association. In addition, we have developed mutagenic strategies to improve the association of engineered, full λετ γη α and β chains.

Supported by grants from NIH.
Blockade of hyaluronan, a CD44 ligand, inhibits IL-2–induced vascular leak syndrome while maintaining effective treatment of melanoma (48.30)
Authors: Guan Hongbing , Nagarkatti Prakash S , Nagarkatti Mitzi
Published: January 2023

High-dose IL-2 therapy is an effective mode of treatment against melanomas; however, its use is limited by accompaniment of vascular leak syndrome (VLS), a life-threatening toxicity. The precise mechanism/s of VLS is unclear and there is no specific therapy to block VLS and enhance IL-2 based immunotherapy. Previous studies from our laboratory demonstrated that CD44 plays a critical role in VLS; administration of hyaluronan (HA), a primary ligand for CD44, caused a marked increase in IL-2 induced VLS. In this study, we report a novel means to prevent VLS by blocking endogenous HA with HA-specific binding peptide, Pep-1. Our results demonstrated that administration of Pep-1 into IL-2 treated mice dramatically inhibited IL-2-induced VLS in the lungs and liver. Interestingly, Pep-1 treatment did not affect the effectiveness of IL-2 in treatment of melanoma. Pep-1 treatment did not affect the emigration of lymphocytes across the endothelium and the activation of CTL seen during IL-2 treatment. Also, blocking HA with Pep-1 maintained endothelial integrity and prevented their apoptosis. Our results demonstrate that HA plays a role in regulating endothelial damage and blockade of HA could be a novel means for prevention and treatment of VLS thereby facilitating IL-2-induced immunotherapy of melanomas.

(Supported by NIH grants RO1 AI 053703, RO1 DA 016545, RO1 AI 058300, RO1 HL 058641 and RO1 ES 09098)
Adenovirus-human HER2 vaccine inhibits breast cancer growth and vaccine induced antibodies (VIA) are efficacious against Herceptin-refractory human breast cancer (48.32)
Authors: Wei Junping , Hartman Zachary , Osada Takuya , Yang Xiao Yi , Lei Gangjun , Jiang Haixiang , Chong Gabriel , Amalfitano Andrea , Lyerly H. Kim , Morse Michael , Clay Timothy
Published: January 2023

While HER2/neu specific antibody (Herceptin) therapy has clear efficacy against HER2 overexpressing tumors, most patients who initially respond to Herceptin show disease progression within one year despite continued high expression of HER2. As an alternative to Herceptin, we developed a novel set of adenoviral vectors encoding human HER2 which break tolerance and induce T and B cell responses in human-HER2-transgenic (HuHER2-Tg) mice (kindly provided by W-Z. Wei, Karmanos Cancer Center, MI). Ad-HER2 vaccination results in a high level of HER2-specific T cells and anti-HER2 vaccine induced antibodies (VIA), which significantly inhibits the growth of human HER2 expressing murine 4T1 breast cancer cells (kindly provided by M. Kershaw, Peter MacCallum Cancer Centre, Melbourne, Australia) in HuHER2-Tg mice. Anti-HER2 VIA strongly bound to Herceptin-refractory human breast tumor cells and significantly inhibited their growth in vitro, and mediated complement dependent cytotoxicity. Thus, our findings demonstrate that Ad-HER2 vaccination proved to be efficacious and therapeutically relevant in Herceptin refractory tumor models.

Grant support: NIH P50-CA068438 SPORE in Breast Cancer & DOD CTRA BC050221.
Friend or Foe? IFNγ Mediates Pro-Metastatic Gene Expression in the Tumor Microenvironment (48.34)
Authors: Hall Veronica L , Subleski Jeff , Back Tim C , Gruys M. Eilene , Shorts-Cary Lynnette , Weiss Jonathan M. , Wiltrout Robert H
Published: January 2023

Several immunotherapeutic models have shown that interferon gamma (IFNγ) is required for tumor regression. Treatment with IFNγ as a sole therapy, however, has resulted in disappointing outcomes. Cytokine therapies, such as IL-2/IL-12 and IL-12/IL-18, each involves IFN-γ mediated reduction in primary tumors but they can have an unanticipated increase in secondary metastases. Therefore, tumor responsiveness to IFNγ may alter its progression and metastasic potential. Mouse renal carcinoma cells expressing a dominant negative IFNγ-receptor had a reduced ability to metastasize, suggesting that IFNγ may exert potentially pro-metastatic effects in the tumor microenvironment. The goal of this study is to identify changes in gene expression in the tumor microenvironment in response to IFNγ treatment. Using qPCR gene profiling, we identified the increased expression of several pro-metastatic genes specifically in the tumor microenvironment after IFNγ treatment, namely heparanase, CD44, s100a4, and matrix metalloproteinase 7. We are now investigating whether the upregulation of these genes is caused by host and/or tumor responses to IFNγ. Our results suggest that IFNγ regulates extra-cellular matrix remodeling protein expression in the local microenvironment, which may contribute to tumor metastases.
Activity of Anti-PD-1 in Murine Tumor Models: Role of “Host” PD-L1 and Synergistic Effect of Anti-PD-1 and Anti-CTLA-4 (48.37)
Authors: Korman Alan , Chen Bing , Wang Changyu , Wu Linda , Cardarelli Pina , Selby Mark
Published: January 2023

PD-1, like CTLA-4, is a negative regulator of T cell function. The PD-1 ligand, PD-L1, is expressed by dendritic cells/antigen presenting cells and by activated T cells. Many tumors express PD-L1 and its expression has been linked to poor prognosis. PD-L1 expression is also upregulated by γIFN. We studied the activity of anti-PD-1 in two subcutaneous tumor models which differ with regard to PD-L1 expression: Sa1N fibrosarcoma, which is negative for PD-L1, and cannot be induced to express PD-L1 by γIFN; and, MC38 colon adenocarcinoma, which expresses low levels of PD-L1 which is upregulated by γIFN. Anti-PD-1 blockade resulted in partial tumor growth inhibition in both models demonstrating that the activity of anti-PD-1 is independent of PD-L1 expression on the tumor. This suggests that host PD-L1 (and/or PD-L2) expression limits anti-tumor activity. In the MC38 model, the concurrent use of anti-PD-1 and anti-CTLA-4 antibodies was significantly more potent than either of the individual antibodies or sequential administration of the antibodies regardless of order. These results are consistent with a role for PD-1 and CTLA-4 in T cell priming and suggest that they play non-redundant roles during T cell activation. Cell subsets implicated in the activity of PD-1 and CTLA-4 blockade in the MC38 model are under investigation.
Selective metalloproteinase inhibition of human CD30 shedding increases specificity of targeted immunotherapy (48.38)
Authors: Hansen Hinrich Peter , Simhadri Vijaya Lakshmi , Eichenauer Dennis , Ludwig Andreas , Engert Andreas , von Strandmann Elke Pogge
Published: January 2023

The receptor CD30 is selectively overexpressed on many human lymphoma cells and therefore an interesting target for antibody-based immunotherapy. However, binding of therapeutic antibodies stimulates CD30 shedding leading to a loss of target antigen and a release of the soluble ectodomain (sCD30). Here, we showed that sCD30 binds to membrane-anchored CD30 ligand (CD153) on mast cells and neutrophils which are frequently found among the bystander cells in lymphoma tissue. Using sCD30 as a linker, CD30 antibodies are able to bind to mast cells where they caused the release of interleukin-8 (IL-8) which is involved in angiogenesis and metastasis. To overcome this adverse shedding-dependent mistargeting we used loss-of-function experiments with cells lacking candidate CD30-releasing enzymes ADAM10 and ADAM17 and a selective inhibitor to identify ADAM10 as the main enzyme responsible for antibody-stimulated shedding. In co-culture experiments, the antibody-induced transfer of sCD30 from the human Hodgkin lymphoma cell line L540 to the CD30-negative but CD153-expressing human mast cell line HMC-1 was inhibited by the ADAM10-selective inhibitor GI254023X. These findings suggest that selective metalloproteinase inhibitors blocking antibody-induced shedding of target antigens could be of therapeutic value to increase the specificity and reduce side-effects of immunotherapy with monoclonal antibodies.
Overexpressed circulating serine proteases are a predominant source of self-antigen for potent T lymphocyte immunity to human leukemia (48.5)
Authors: Sergeeva Anna , Ono Yoko , Molldrem Jeffrey J
Published: January 2023

From the experience of clinical transplantation, cytotoxic T lymphocytes (CTL) mediate strong anti-leukemia immunity, although a little is known about the targets of that immunity. We identified an HLA-A2- restricted nonomer PR1 (VLQELNVTV) from proteinase 3 (P3) and neutrophil elastase (NE) that was recognized by CTL, that preferentially killed leukemia cells. To determine the protein source of PR1, we studied distribution of both P3 and NE in patients with AML and CML. Serum NE (ng/ml) assessed by ELISA was increased in 13 out of 14 AML patients and in 19 out of 20 CML patients, compared to that in 4 healthy donors (HD) (1055 ± 132, and 1002 ± 103 versus 54 ± 13; p = 0.0002 and p = 0.0006, respectively). In contrast, overexpression of serum P3 (ng/ml) in 19 AML patients and 28 CML patients was not significantly different compared to the mean from 14 HD (33 ±18 and 56 ± 21 versus 10 ± 2.2; p = 0.13 and p = 0.08, respectively). In nine myeloid leukemia patients the marrow-derived P3 level, which was similar to serum P3, was also similar to that in five non-myeloid leukemia patients (23 ± 9.5 versus 27 ± 11.8, p = 0.08). Further P3 was highly expressed on plasma membrane (PM) of both marrow-derived and circulating blasts, whereas NE was only expressed on PM of marrow-derived blasts. Therefore aberrant expression of serine proteases in leukemia may play a role in reversing T cell tolerance by increasing available determinants on MHC-I in leukemia.
Breast Cancer Peptide Epitopes Presented by HLA-A*0201 (48.6)
Authors: Hawkins Oriana E , VanGundy Rodney , Fleshman Annette , Bardet Wilfried , Gonzalez Melva , Wahl Angela , Buchli Rico , Hildebrand William
Published: January 2023

Cellular immune mechanisms detect and destroy cancerous and infected cells via the human leukocyte antigen (HLA) class I molecules that present peptides of intracellular origin on the surface of all nucleated cells. The identification of novel, tumor-specific epitopes is a critical step in the development of TCR mediated immunotherapeutics. In order to directly identify peptide epitopes unique to cancerous cells, secreted human class I HLA molecules were constructed by deletion of the transmembrane and cytoplasmic domain of HLA A*0201. The resulting sHLA-A*0201 was transferred and expressed in breast cancer cell lines MCF-7, MDA-MB-231, and BT-20 as well as in the immortal, non-tumorigenic cell line MCF10A. Stable transfectants producing sHLA-A*0201 were seeded into Bioreactors for production of > 25 mg of sHLA-A*0201. Peptides eluted from affinity purified sHLA were analyzed by mass spectroscopy and peptides derived from proteins with reported cancer association have been identified; BAP31, Cytokeratin 19, KIAA0336, and UGT1 peptide epitopes have been found on cancer cells. The identification of peptides unique to the class I of cancerous breast cells provides putative targets for immune diagnostics and therapeutics.

OH is supported by NSF Graduate Fellowship 2006036207
A Novel Immune-Based Cancer Therapy Using Gene-Silenced Dendritic Cells (48.8)
Authors: Zheng Xiufen , Zhang Xusheng , Vladau Costin , Li Mu , Suzuki Motohiko , Chen Dong , Garcia Bertha , Koropatnick James , Min Wei-Ping
Published: January 2023

Hyporesponsiveness is a major hallmark in dendritic cells (DC)-mediated anticancer immunotherapy. Indoleamine 2, 3-dioxygenase (IDO), an immunosuppressive molecule expressed by DC, is a critical factor mediating hyporesponsiveness to cancer immune therapy. We hypothesized that silencing of IDO in DC using siRNA would enhance anticancer therapy. In this study, DC were cultured in vitro, exposed to melanoma B16 lysate, silenced with IDO siRNA, and injected into C57/BL6 mice. Mice were then challenged with B16 tumor cells. The anticancer effects of IDO-silenced DC therapy, compared with non-silenced, conventional control DC vaccine, were evidenced by

postponed melanoma tumor onset time, anddecreased tumor size.

In addition, after immunization with IDO-silenced DC, the number of CD8+ T cells was significantly increased while CD4+ and CD8+ T cell apoptosis in draining lymph nodes was remarkably reduced. Furthermore, immunization with IDO-silenced DC enhanced tumor antigen-specific T cell proliferation and CTL activity, and decreased numbers of CD4+CD25+FoxP3+ regulatory T cells (Treg). In conclusion, this study is the first to demonstrate a novel anti-tumor vaccine by silencing an immunosuppressive gene (IDO) in DC, which enhanced anti-tumor immunity, reduced T cell apoptosis and Treg cell formation, and prevented tumor growth. IDO-silenced DC have clinical potential as an immune-based therapy.
CCL2 producton in neutrophils (PMN) from peripheral blood of hepatocellular carcinoma patients (49.1)
Authors: Tsuda Yasuhiro , Asai Akira , Fukui Hideo , Tanabe Toshiaki , Teramura Kazuhisa , Fukuda Akira , Hanafusa Toshiaki
Published: January 2023

An immunosuppressive subset of PMN has recently been demonstrated in mice with severe immunocompromised status such as severe systemic inflammatory response syndrome (SIRS), severe thermal injury or severe stress. By CCL2 production, these PMN direct resident macrophages (MƒÓ) to M2MƒÓ. Functional deficiencies of tumor-associated immune cells have been shown in cancer patients bearing acceleration of tumor growth who are known to be immunocompromised hosts. Hepatocellular carcinoma (HCC) was one of a most frequent malignant tumor in worldwide. Repeated episodes of recurrence and metastasis are commonly associated with the poor prognosis of HCC. Therefore, in this study, subsets of PMN from HCC patients were immunobiologically investigated. Thirty HCC patients admitted to Osaka Medical College were randomly enrolled. PMNs were isolated from heparinized peripheral blood using Ficoll-Hypaque and dextran sedimentation. PMNs from 5 healthy donors were used as controls. PMN (1 x 106 cells/ml) were stimulated with 0.0075% SAC for 24 hrs. Culture fluids harvested were assayed for CCL2 by ELISA. After SAC stimulation, CCL2 were produced by all patient PMNs, while none of healthy donor PMNs produced these soluble factors. Moreover, CCL2 production was significantly increased in proportion to tumor size or extent. These results suggest that PMN with a function to produce CCL2 are predominated in HCC patients and they may be involved in their poor prognosis.
Expansion of Spleen Myeloid Suppressor Cells Represses NK Cell Cytotoxicity in Tumor Bearing Host (49.13)
Authors: Liu Cunren , Yu Shaohua , Zinn Kurt , Wang Jianhua , Grizzle William E , Kappes John , Zhang Huang-Ge
Published: January 2023

Tumor growth promotes the expansion of myeloid suppressor cells. An inverse correlation between natural killer (NK) cell activation and myeloid suppressor cell expansion in tumor-bearing patients and mice prompted us to investigate the role of myeloid suppressor cells in controlling NK cell antitumor cytotocixity. In TS/A breast tumor model, the in vivo NK cell cytotoxicity was impaired in tumor-bearing mice. After adoptive transfer to naive recipients, CD11b+Gr-1+ myeloid suppressor cells freshly isolated from spleens of tumor-bearing mice but not naive mice were able to inhibit NK cell cytotoxicity. An in vivo imaging analysis indicated that the removal of tumors resulted in a significant increase in NK cell cytotoxicity in lung to eliminate injected YAC-1 cells. FACS analysis of the composition of lung leucocytes further indicated that the removal of tumors also lead to the reduction of CD11b+Gr-1+ myeloid suppressor cells accumulated in the lung. These data suggest that CD11b+Gr-1+ myeloid suppressor cells suppress NK cell cytotoxicity. The inhibition of NK cell cytotoxicity is cell-cell contact dependent. Inhibition of perforin but not granzyme B was responsible for myeloid suppressor cell-mediated inhibition of NK cell cytotoxicity. Western blot analyses further suggested that myeloid suppressor cells suppress IL-2 mediated NK cell cytotoxicity by affecting the activity of Stat5.
Tumor exosomes inhibit differentiation of bone marrow dendritic cells (49.14)
Authors: YU Shaohua , Liu cunren , Wang Jianhua , Liu yuelong , Zhang liming , Cong Yingzi , Grizzle william , Zhang huang-Ge
Published: January 2023

The production of exosomes by tumor cells has been implicated in tumor-associated immune suppression. Here we show that, in mice, exosomes produced by TS/A murine mammary tumor cells target CD11b+Gr-1+ myeloid precursors in the bone marrow in vivo and that this is associated with an accumulation of myeloid precursors in the spleen. Moreover, we demonstrate that TS/A exosomes block differentiation of murine myeloid precursor cells into dendritic cells in vitro. Addition of tumor exosomes at day 0 led to a complete block of differentiation into dendritic cells, whereas addition at later time points was less effective. Similarly, exosomes produced by human breast tumor cells inhibited differentiation of human monocytes in vitro. The levels of IL-6 and phosphorylated Stat3 were elevated 12 h after tumor exosome stimulation of murine myeloid precursors, and tumor exosomes were less effective in inhibiting differentiation of bone marrow cells isolated from IL-6 knockout mice. These data suggest that tumor exosome-mediated induction of IL-6 plays a role in blocking bone marrow dendritic cell differentiation.
Downstream effects of decreased microRNA miR-16 levels, found in NZB B cell malignancies, include alterations in cell cycle progression and apoptosis (49.23)
Authors: Salerno Erica , Scaglione Brian , Balan Murugabaskar , Kotenko Sergei V , Coffman Frederick , Raveche Elizabeth
Published: January 2023

Alterations in the 13q14 genomic region containing mir-15a and mir-16-1 have been found in human CLL, resulting in down-regulation of these miRNAs in some patients. NZB mice, serving as a model for CLL, were used to study this particular region on chromosome 14, which shows synteny with the human 13q14 region. We recently identified a point mutation in the 3′flanking region of mir-16-1, similar to the one found in human CLL, that may be associated with decreased levels of miR-16. Decreased miR-16 can result in increased expression of miR-16 target genes, such as bcl-2, leading to failure of tumor cells to undergo apoptosis. When cells from an NZB-derived malignant B cell line are transfected with exogenous miR-16 at various phases of the cell cycle an increase in apoptosis as well as various cell cycle effects can be observed. Cells in or entering S phase remain in S phase longer when treated with miR-16. In summary, alterations in the levels of miR-16 in the NZB model, resulting from mutations in the 3′flanking region, may be associated with changes in cell cycle transit and apoptosis induction. These findings suggest that miR-16 plays a role in the development and progression of B cell malignancies.
Vitamin C potentiates gefitinib-mediated tumor cell growth inhibition in human lung cancer cells (49.26)
Authors: Bae Seyeon , Hahm Eunsil , Lee Kyung Eun , Kang Jae Seung , Jung Da Jung , Kim Jee Eun , Lee Seung Koo , Hwang Young Il , Shin Dong Hoon , Lee Wang Jae
Published: January 2023

Malignancies of lung are the leading causes of cancer mortality in world wide and there are so many limitations in early detection of lung cancer, which eventually made the cancer detected at advanced stage. Conventional cytotoxic drugs have showed more increased response rate with grave toxicities. Recently, successful therapy with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor in lung cancer patients have been reported, although a mono-therapy still showed low response rate. Here, we investigated to elucidate whether the vitamin C has the adjuvant role as an antioxidant or other functions in EGFR tyrosine kinase inhibitor, gefitinib (Iressa ¢â), therapy in lung cancers. We used three kinds of human non-small cell lung cancer cell (NSCLC) lines depending on EGFR mutation, A549, Calu-3 and HCC827. A549 and Calu-3 are NSCLC cell lines which have wild type EGFR domain and showed relative resistance to gefitinib treatment. HCC827 is well known cell line of EGFR domain mutation. Vitamin C showed reduction of S phase in cell cycle and had function as an anti-oxidant. Also that could induce synergistic effects of EGFR tyrosine kinase inhibitor therapy in cellular proliferation and down-regulation of EGFR phosphorylation, AKT & ERK activation. Synergistic inhibitory effects of vitamin C with EGFR tyrosine kinase inhibitor therapy are showed in both wild and mutant EGFR cell lines. In this present study, we demonstrated an possible adjuvant role of vitamin C in EGFR tyrosine kinase inhibitor therapy in human NSCLC.
Expression of Vascular Endothelial Growth Factor Receptor 1(VEGFR-1), but not VEGFR-2 in Activated T Lymphocytes and Its Function in Response to VEGF (49.28)
Authors: Shin Jin Young , Kim Jung Sik , Kim Bongi , Yun Il Hee , Park Chung Gyu
Published: January 2023

Vascular endothelial growth factor (VEGF) is the most potent proangiogenic mediator that plays an important role in tumor growth and progression. Recent evidence suggests an alternate role of VEGF and its receptor 1 and 2 (Flt-1 and Flk-1) on host immune response to tumors. Activated or effector T lymphocytes are the chief type of defense cells mediating cellular immunity against tumor cells. However, expression of VEGF receptors in activated T lymphocytes is not yet identified and its function is unclear. We have studied expression of VEGF receptors, VEGFR-1 and VEGFR-2 in these cells. Activated T lymphocytes express mRNA for VEGFR-1 and R-2. But these cells are found to express the protein for VEGFR-1, which is one of the receptors for VEGF. We demonstrate that treatment with VEGF directly induced the production of IL-10 with slight reduction of IFN-γ although it dose not affect T cell proliferation. A chemotaxis assay reveals that activated T lymphocytes have the ability to migrate in response to VEGF. Our data suggest that in the presence of VEGF, VEGFR1 positive-activated T lymphocytes may migrate to tumor sites and be involved in tumor tolerance through IL-10
Expression and functions of Xg protein on Ewing’s sarcoma cells (49.29)
Authors: Meynet Ophelie , Marti Virginie , Scotlandi Katia , Bernard Alain , Bernard Ghislaine
Published: January 2023

The human Xg molecule is a 26 kD transmembrane glycoprotein normally displayed by red cells only. It shows 48 % of homology with CD99, known as a regulator of cellular adhesion and migration. Xg is encoded by the PBDX gene located on the pseudoautosomal region of X chromosome. To determine the functional role of Xg we have developed a Jurkat T cell model transfected with the Xg cDNA. Xg positive Jurkat T cells spontaneously display a strongly activated cytoskeleton with extensive actin polymerization, microvilli formation and strong aggregation. Moreover, this was accompanied by an increase migratory ability upon SDF-1 chemoattraction.

Unexpectedly, we found Xg to be expressed by malignant cells and particularly by 80% of Ewing sarcomas cell lines. We have transfected the Xg cDNA in Xg negative Ewing’s sarcoma cell line BRZ which induced similar activities as those we found for transfected Jurkat T cells. It must be now looked whether Xg expression like CD99 expression is restricted to ES cells among sarcoma cells is linked to an invasive phenotype.
Inhibition of Bence-Jones protein synthesis by RNA interference (49.30)
Authors: Phipps Jonathan Earl , Foster James Stephen , Kestler Daniel , Alan Solomon , Wall Jonathan Stuart
Published: January 2023

A main contributing factor to the morbidity and mortality of patients with multiple myeloma results from the pathologic deposition as casts within renal tubules of monoclonal Ig light chains, i.e., Bence-Jones proteins (BJP). As an alternative to plasma cell chemotherapy to reduce BJP synthesis and thus prevent myeloma (cast) nephropathy we have investigated another means to achieve this objective, namely, RNA interference (RNAi). Experimentally, we have stably transfected the SP2/O mouse myeloma cells with a construct containing the V, J, and C region of a λ BJP (Wil) under the control of a CMV promoter and have shown that they constitutively express measurable quantities of mRNA and protein Wil. Treatment of these cells with synthetic, small double stranded RNA based on the Wil protein sequence reduced target mRNA levels by 2-fold at 48 h and protein production by 30% at 72 h, as compared with controls. These data provide conclusive evidence that RNAi can effectively reduce BJP production in vitro and provide the base for testing the clinical potential of this strategy using a relevant in vivo murine model of myeloma (cast) nephropathy.
Pre-operative interleukin-6 production by mononuclear blood cells predicts survival after radical surgery for colorectal carcinoma (49.31)
Authors: Clinchy Birgitta , Fransson Annelie , Druvefors Bengt , Hellsten Anna , Håkansson Annika , Gustafsson Bertil , Sjödahl Rune , Håkansson Leif
Published: January 2023

Colorectal cancer (CRC) is one of the most common forms of cancer in the Western world with an estimated yearly incidence rate of 77 per 100 000 in North America. Besides histopathological staging, there is a great need for tests giving more accurate prognostic information defining the patient population in need of closer follow-up and/or adjuvant therapy. Elevated serum levels of IL-6 have been found in patients with CRC. The source of the serum IL-6 is still unclear.

Peripheral blood mononuclear cells (PBMC) were isolated preoperatively (within a week before surgery) from 34 patients undergoing radical surgery for colorectal cancer. The PBMCs were cultured for 24 hours in the presence of autologous serum and LPS. IL-6 production was measured with ELISA. Staging based on histopathology was performed in all patients.

A production of >5000 pg/ml of IL-6 identified CRC patients with a poor prognosis. Eight out of thirteen patients with >5000 pg/ml IL-6 died from cancer within the follow-up period (at least 54 months) whereas no cancer related deaths were recorded among twenty-one patient with 5000 pg/ml IL-6 or less. IL-6 production was identified as an independent prognostic factor. These data support the hypothesis that the adaptive immune system is of importance for tumour progression and that immunological parameters can serve as useful as prognostic and predictive tools in cancer management.
Tumor modulation of T-helper cytokine secretion (49.6)
Authors: Lathers Deanne M.R. , Kearney Patricia L. , Gibbs Kiki , Young M. Rita
Published: January 2023

Tumor modulation of T-cells contributes to tumor-induced immune suppression. To determine if tumor-derived factors selectively recruit T-cell subpopulations, the migration of T-cells in response to SCC VII/SF or epithelial cell (epi)-conditioned media (CM) was determined by Transwell chemotaxis assay. Increased levels of CD4+CD25+ cells were observed in T-cells migrating in response to SCC-CM. Following stimulation, T-cells migrating in response to SCC-CM had diminished IFN-γ secretion as compared to epi-CM. Additional studies determined whether SCC-CM actively suppresses IFN-γ secretion by Th1 cells in the tumor microenvironment. Th1 and Th2 cells were differentiated from naïve lymph node cells then cultured with SCC or epi-CM. After overnight stimulation, IFN-γ and IL-10 secretion was assessed by ELISA. Following SCC-CM treatment, IFN-γ secretion by Th1 cells was diminished as compared to epi-CM treatment. IL-10 secretion by Th2 cells treated with either CM was unaffected. The effects of SCC-CM on IFN-inducible chemokines are currently under investigation. These results identify mechanisms of tumor-induced immune suppression by demonstrating that Th2 cells are selectively recruited in response to SCC-derived factors and that Th1 cells in the tumor microenvironment have suppressed IFN-γ secretion.

Supported by Dept. of Veterans Affairs, American Cancer Society and National Cancer Institute.
The role of paracrine IL-6 and IL-10 in neuroblastoma progression (49.9)
Authors: Song Liping , Ara Tasnim , Salo Jill , Deng Zhong-bin , Asgharzadeh Shahab , Ji Lingyun , Sposto Richard , DeClerck Yves A , Seeger Robert C , Metelitsa Leonid S
Published: January 2023

Tumor-associated inflammation may play a critical role in tumor progression but its mechanism is yet poorly understood. We have detected elevated levels of IL-6 (2.9 – 88.9 pg/ml) in serum from 10 patients with recurrent stage 4 neuroblastoma. Although none of 5 tested neuroblastoma cell lines produced either IL-6 or IL-10, their conditioned medium selectively induced both cytokines in short-term culture of normal PBMC in a time- and dose-dependent manner as determined by 9-plex Luminex® assay. Treatment with rhIL-6 enhanced neuroblastoma cell proliferation as measured by BrdU incorporation and was sufficient for their survival in a serum-free medium. IL-6 induced rapid STAT3 phosphorylation in neuroblastoma cells and production of yet unidentified soluble factors that inhibited dendritic cell maturation. Gene expression analysis with TaqMan® Low Density Arrays of primary metastatic tumors from 40 patients revealed that elevated expression of IL-6 or IL-10 was associated with a poor 5-year event-free survival: 35% +/−11% vs. 75 +/−10% when IL-6 or IL-10 expression was above and below median levels, respectively (P < 0.02). Thus, although neuroblastoma cells do not express IL-6 and IL-10, they can induce expression of these cytokines by inflammatory cells. These cytokines can then have a paracrine effect on tumor cells and other elements of tumor microenvironment that may promote tumor progression.
Murine L5178Y T-cell lymphoma cells prime macrophages via CD40-CD40L interaction to mediate LPS-activated antitumor effects in vitro (50.13)
Authors: Buhtoiarov Ilia N. , Rakhmilevich Alexander L. , Sondel Paul M.
Published: January 2023

We present that L5178Y murine T-cell lymphoma cells can prime naïve murine macrophages (Mϕ) to subsequent LPS stimulation, resulting in antitumor effects in vitro against L5178Y cells. Priming occurred in both cell-type and TLR specific manner, as L5178Y tumor cells (TC), but not naïve splenocytes, primed Mϕ to ligation of TLR4 but not TLR9. L5178Y-primed Mϕ also showed down-regulation of CD40 and CD86 and up-regulation of B7H1, with no changes of TLR4, B7H4, NKG2D and MHC-II expression. Among different TC lines, the priming effect was limited to murine TC of T cell (L5178Y and YAC-1) but not B cell (A20) or nonmyeloid (B16) origin. Human TC lines including Jurkat (T cell), Daudi (B cell), and M21 (melanoma) failed to substantially prime Mϕ. Neither L5178Y-conditioned supernatants nor co-culture of Mϕ and L5178Y TC in transwells resulted in priming, indicating that direct L5178Y TC-Mϕ contact was needed. Furthermore, TLR4-ligation must not precede contact with L5178Y TC in order to induce tumoritoxic Mϕ. Among several receptor-ligand pairs reciprocally expressed on Mϕ and L5178Y TC and potentially involved in the Mϕ priming, CD40 (expressed on Mϕ) and CD40L (expressed on L5178Y TC) appeared to play the most important role, as blocking their interaction with anti-CD40L mAb substantially reduced in vitro tumoritoxicity. Hence, contact with TC may facilitate Mϕ-mediated immune surveillance against certain cancers.
Anti-B7-1/B7-2 mAb treatment enhanced anti-tumor immunity in TRAMP mice (50.14)
Authors: Zheng Pan , Zhou Penghui , Zheng Xincheng , Zhang Huiming , Liu Yang
Published: January 2023

Immune tolerance is the major mechanism applied by the immune system to discriminate between self and non-self. There is sufficient evidence to show that CD4+CD25+ Treg cells play an important role in the maintenance of peripheral tolerance. Accumulating evidence suggests that CD4+CD25+ Treg cells also suppress tumor antigen-specific immunity in tumor-bearing mice and in humans. Therefore, breaking Treg-mediated peripheral tolerance to tumor antigens may enhance anti-tumor immunity. B7:CD28 interactions are needed for both the development and maintenance of CD4+CD25+ Treg cells. This raises the possibility that blocking the B7 signal may break tumor tolerance by reducing the number of CD4+CD25+ Treg cells. Here we show that administration of anti B7-1/B7-2 mAbs either at a beginning stage or at an intermediate stage of prostate cancer development significantly enhanced anti-tumor immunity by postponing the disease process. Anti-tumor immunity was improved after inhibiting the generation of CD4+CD25+ regulatory T cells in thymus and periphery without a hint of autoimmune disease. Our results demonstrate that blocking the costimulatory pathway with anti B7-1/B7-2 antibodies can enhance anti-tumor immunity.
Type II NKT cells suppress tumor immunosurveillance enhanced by type I NKT cells (50.16)
Authors: Terabe Masaki , Ambrosino Elena , Takaku Shun , Peng Judy , Miyake Sachiko , Halder Ramesh , Yamamura Takashi , Kumar Vipin , Berzofsky Jay
Published: January 2023

NKT cells are a T cell subset that recognizes glycolipid antigens presented by CD1d. They consist of 2 subsets: the widely studied type I expressing a canonical TCR, Vα14Jα18, and a little known type II of non-Vα14Jα18 NKT cells. Here, we examined the role of type I and II NKT cells in the regulation of tumor immunosurveillance. Jα18 KO mice, which lack type I but retain type II NKT cells, had a significantly larger tumor burden of CT26 tumor injected i.v. than wild-type mice early in tumor growth. This result suggested that the absence of type I NKT cells allows type II NKT cells to further suppress tumor immunosurveillance compared to wild-type mice that have both types of NKT cells. The role of NKT cell subsets was examined by stimulating them with antigens that specifically activate either type I or type II NKT cells. Mice treated with type I NKT cell agonist α-galactosylceramide or the weaker agonist OCH almost completely rejected tumor. In contrast, mice treated with a single dose of type II NKT cell-stimulating sulfatide had significantly increased tumor burden. Simultaneous stimulation of both types of NKT cells in vivo significantly suppressed the protective effect of type I NKT cell stimulation. Overall, the results suggested that stimulation of type I NKT cells promotes tumor immunosurveillance, whereas stimulation of type II NKT cells suppresses, and that type II NKT cells suppress type I NKT cell activity.
Targeting molecular and cellular inhibitory mechanisms for improvement in anti-tumor memory responses reactivated by tumor cell vaccine (50.17)
Authors: Dong Haidong
Published: January 2023

Development of effective vaccination approaches to treat established tumors represents a focus of intensive research because such approaches offer the promise of enhancing immune system priming against tumor antigens via re-stimulation of pre-existing (memory) antitumoral helper and effector immune cells. However, inhibitory mechanisms which function to limit the recall responses of tumor-specific immunity exist remain poorly understood and interfere with therapies anticipated to induce protective immunity. The mouse renal cell carcinoma (RENCA) tumor model was used to investigate variables affecting vaccination outcomes. We demonstrate that although a whole cell irradiated tumor cell vaccine can trigger a functional anti-tumor memory response in the bone marrows of mice with established tumors, these responses do not culminate in the regression of established tumors. In addition, a CD103+ regulatory T (Treg) cell subset accumulates within the draining lymph nodes of tumor-bearing mice. We also show that B7-H1 (CD274, PD-L1), a negative costimulatory ligand, and CD4+ Treg cells collaborate to impair the recall responses of tumor-specific memory T cells. Specifically, mice bearing large established RENCA tumors were treated with tumor cell vaccination in combination with B7-H1 blockade and CD4+ T cell depletion (triple therapy treatment) and monitored for tumor growth and survivability. Triple treatment therapy induced complete regression of large established RENCA tumors and raised long-lasting protective immunity. These results have implications for developing clinical antitumoral vaccination regimens in the setting where tumors express elevated levels of B7-H1 in the presence of abundant Treg cells.