bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of Antigen-Specific CD8+ T cells in Melanoma Patients using a PrimeBoost Therapuetic Vaccine (48.18)
Authors: Anderson Richard John , Whale Kevin , Brown Dean , Scheibenbogen Carmen , Asemissen Anne Marie , Cripps Martin , Schneider Joerg ,   The Hi-8 MEL Phase I/II Study Group
Published: January 2023

Induction of tumour-specific CD8+ T cell responses in melanoma patients is the major aim of antigen-specific immunotherapy. This study evaluated a prime-boost immunotherapeutic vaccine (Hi-8™ MEL) in stage III/IV melanoma patients using vectors encoding a polyepitope string of seven CD8+ T cell epitopes from five melanoma antigens. T cell responses were evaluated in cryopreserved PBMC samples taken before and during treatment. CD8+ T cells specific for the immunodominant Melan-A epitope were detected by ex vivo tetramer assay in 24/36 (67%) of all patients and in 10/11 (91%) of patients that received the highest treatment dose. Eleven of 36 (31%) patients mounted an ex vivo IFN-γ ELISPOT response: 9/36 (25%) to Melan-A and 3/36 (8%) to other epitopes in the string (NY-ESO-1 and MAGE-3). Notably, the Melan-A-tetramer+ CD8+ T cells in one patient reached 7.25% and acquired a CD45RA−CCR7− effector/memory phenotype. This patient also exhibited an ELISPOT response and a partial clinical response (>12 months). Across the study, tetramer responders showed a median survival of 86 weeks versus 37 weeks for non-responders (p<0.001), indicating that Hi-8™ MEL immunotherapy can elicit melanoma-specific CD8+ T cell responses that are associated with tumour control and increased survival. Additional immune assays are underway to further elucidate the nature of these efficacious T cell responses.
HLA-B-Associated Transcript 3 (BAT3) is an activating ligand for NKp30 and released from dendritic cells (48.2)
Authors: Simhadri Venkateswara Rao , Reiners Katrin S , McKinnon Peter J , Hallek Michael , Engert Andreas , von Strandmann Elke Pogge
Published: January 2023

Natural-killer (NK)-cells, originally defined as innate lymphocytes are regulators of adaptive immune responses via cytokine secretion and cross-talk with dendritic cells (DCs).

Both killing and maturation of DCs by NK cells is dependent on NKp30, a Natural Cytotoxicity Receptor (NCR) expressed on NK cells. However, the NKp30-specific ligands expressed on dendritic cells have remained elusive.

We recently identified BAT3 as the tumor associated ligand for NKp30. BAT3 is a nuclear protein characterized by the conserved BAG domain (Bcl-2 associated anthogene), that interacts with HSP70. Here we show that immature and mature DCs express BAT3 mRNA and protein. The protein is released from DCs into the extracellular environment upon exposure to stress signals such as serum starvation and heat shock. Moreover, the purified, recombinant BAT3 is functional as it binds to NKp30 and induces NK cell-mediated cytokine secretion. The inhibition of BAT3 using polyclonal antibodies prevents tumor rejection in vivo, demonstrating that BAT3 is necessary for the tumor rejection in a xenograft tumor model.

Thus, BAT3 is a novel ligand for the NK cell-receptor NKp30 and its role in the reciprocal activation of NK-DC cross-talk will be discussed. We present evidence that BAT3 is, together with HSP70 and HMGB1, a member of the emerging group of immune regulators that we call “Nucleokines”.

Support- Koeln Fortune Programme (89/2004).
Biodegradable Artificial Antigen Presenting Cells for ex vivo stimulation of antigen-specific T Lymphocytes (48.9)
Authors: Fahmy Tarek M , Steenblock Erin R
Published: January 2023

While dendritic cells (DCs) are the most potent professional antigen-presenting cells, their widespread utility in therapeutic approaches involving ex vivo stimulation of T lymphocytes is hindered by several practical factors such as generation of autologuous DCs, labor and isolation and preparation for clinical applications.

Thus, artificial antigen-presenting cells (aAPC) have been proposed and tested in the expansion of a number of specific T cells for the treatment of a variety of disease states. Here we have developed a novel physiologically compatible microparticulate system for ex vivo T cell stimulation using biodegradable polymers. Our approach fabricates FDA approved polymers into microparticles presenting T cell antigens, thus providing the multivalent interactions necessary for efficient stimulation of T cells. A key feature of this technology is the ability to co-encapsulate cytokines and program their release during and after activation and in a sustained fashion, facilitating long-term bio- availability and efficient stimulation of lymphocytes.
Circulating Immature Myeloid Cells (ImC) Levels Correlate with Clinical Cancer Stage and Cyclophosphamide (CTX) Treatment (49.15)
Authors: Montero Alberto J. , Salem Mohamed L. , Al-Khami Amir , Vissage Kristi , Diaz-Montero C. Marcela
Published: January 2023

Tumor-associated immune suppression is known to be an important contributor to cancer progression. One such mechanism is the abnormal accumulation of ImC in the peripheral blood. Although both tumor burden and CTX-induced accumulation of ImC are well described in animal studies, human studies have been limited. Thus, the goal of our study was to determine whether levels of ImC in whole blood correlate with clinical cancer stage in cancer patients (pts). Flow cytometry analyses were performed on whole peripheral blood from newly diagnosed solid tumor pts. ImC population was defined as lineage−CD33+HLA-DR−CD11b+. Peripheral blood specimens from 14 normal healthy controls and 35 cancer pts (stages: I, n=3; II, n=9; III, n=9; IV, n=14), revealed greater ImC in cancer pts vs. normal (3.21% vs. 1.47%; p=.002). Stage IV pts had the highest percentage of ImC (4.39%). Initial results from stage II-III breast cancer pts (n=10) treated with preoperative chemotherapy showed that CTX treatment was associated with significant increases in circulating ImC compared with baseline (2.91% vs. 10.57%; p=0.001), and also when compared to taxane chemotherapy independent of G-CSF (10.57% vs. 3.25%; p=0.00025). In conclusion, overall ImC levels correlate with clinical cancer stage and CTX treatment leads to transient increases in ImC in breast cancer pts.
Macrophages from tumor-bearing mice contribute to tumor immune escape: a novel mechanism (49.16)
Authors: Torroella-Kouri Marta , Harhaj Ed , Alonso-Diaz Luis , Carrio Roberto , Cardentey Yoslayma , Lopez Diana M
Published: January 2023

Tumor progression is associated with the development of a state of immunosuppression in the host. Macrophages from mice bearing advanced mammary tumors are profoundly dysfunctional, exhibiting decreased levels of several pro-inflammatory cytokines and of nitric oxide, which are critical in the anti-tumor immune response. We have found that the impaired function of these macrophages is associated with reduced expression and nuclear translocation of transcription factors NFkB and C/EBP. The diminished expression of these transcription factors is observed both at the protein and mRNA levels, and interestingly, there is evidence that proteasomal degradation might also be involved in their proteolysis. We have shown that several factors produced by the mammary tumor are associated with this phenomenon. Our results provide evidence that NFkB and C/EBP are targets of the tumor evasion mechanisms observed in advanced cancers.
Preferential Migration of T Regulatory Cells Mediated by Glioma-Secreted Chemokines (49.3)
Authors: Sun Wei , Jordan Justin T , Hussain Sakina F , DeAngulo Guillermo , Weinberg Jeffery S , Heimberger Amy B
Published: January 2023

Despite the immunogenicity of glioblastoma multiforme (GBM), the most common primary brain neoplasm, immune clearance of these tumors is still insufficient for tumor eradication and control. Elevated frequency of CD4+ CD25+FoxP3+ T regulatory (Treg) cells in peripheral blood or tumor-infiltrating lymphocytes has been observed in patients with various solid tumors including glioblastoma. This presence of high Treg fraction contributes to the dismal immune responses generated within these patients and prompted us to investigate the possible mechanism of Tregs selective recruitment in GBM patients. We hypothesized that the chemokines produced by gliomas induce this preferentially Treg migration. In the human malignant glioma cell lines D-54, U-87, U-251 and LN229, the chemokines CCL22 and CCL2 were preferentially produced compared to CXCL9, CCL3, and CCL4 by using cytokine flow cytometry and confirmed with ELISA. Additionally, gliomas obtained directly from human patients demonstrated a similar chemokine expression profile. Glioma supernatants, recombinant CCL2 and CCL22 could induce the migration of Tregs, which could be blocked by neutralizing antibodies to these chemokines. Flow cytometric investigation of the expressions of the CCL2 and CCL22 chemokine receptors CCR2 and CCR4 on peripheral T cells indicated that GBM patients have more CD4+CD25+ T cells that express CCR2 (26%) and CCR4 (53%) in contrast to the CD4+CD25+ T subset from normal individuals that express CCR2 (17%) and CCR4 (42%). These data indicate that gliomas modulate immunosuppression by the selective recruitment of Tregs into the tumor microenvironment and those therapies that modulate this interaction may be potentially synergistic with tumor immunotherapies.
Role of hepatocyte growth factor in the thymic involution of tumor-bearing mice. (49.32)
Authors: Carrio Roberto , Torroella-Kouri Marta , Lopez Diana M
Published: January 2023

During mammary tumorigenesis, there is a profound thymus involution associated with a severe depletion of the most abundant subset of thymocytes, CD4+CD8+ immature cells, however, the mechanisms involved in this phenomenon remain to be elucidated. We have previously reported that the thymic involution observed in tumor bearing mice is associated with an early block in T cell maturation. This alteration in thymocyte differentiation might be due to an impaired function of stromal cell microenvironment in this organ. Using the D1-DMBA-3 mammary tumor model we found that IL-7 and IL-15 mRNA, crucial cytokines involved in thymic development and cellular homeostasis respectively, are profoundly down-regulated in the thymuses of tumor hosts. Importantly, the expression of the gene for hepatocyte growth factor (HGF), which has been associated with proliferation, cell survival, angiogenesisand differentiation, is down-regulated in thymuses of tumor bearers. Injection of HGF into mice implanted with the mammary tumor resulted in a partial restoration of the thymus volume and of the levels of IL-7 and IL-15 in this organ. Collectively, our data suggest that HGF may play an important role in the thymic involution in tumor bearing hosts and that the cytokines expressed in the thymus microenvironment may be modulated by this growth factor.
Human dialyzable leukocyte extracts (DLE) have ligands for TLR- 2 but not for TLR-4 (49.34)
Authors: ROBLEDO-AVILA FRANK , WONG-BAEZA ISABEL , SERAFIN-LOPEZ JEANET , ISIBASI-ARAUJO ARMANDO , LOPEZ-MACIAS CONSTANTINO , PEREZ-TAPIA SONIA MAYRA , ESTRADA-GARCIA IRIS CITLALLI , ESTRADA-PARRA SERGIO ANTONIO
Published: January 2023

Human dialyzable leukocyte extracts (DLE) have been used for the last 50 years to successfully treat different diseases. They are obtained by disruption of cells in buffy coats and subsequent dialysis (<12 KDa) of the extracts. They can transfer cellular immune response from an immune donor to a naïve recipient, and despite their immunomodulatory effect, until now little is known about their mechanism of action. One possibility is that they may contain ligands that activate TLRs. In this work using a reporter gene assay and HEK293 cells transfected with either TLR-2 or TLR-4 plasmids, we showed that different concentrations of DLE (100, 10 and 1 microg/mL) resulted in the activation of NF-kappaB (luciferase reporter system) in cells TLR-2 transfected cells with 10 or 1 microg/mL DLE. Interesting none of the DLE concentrations used induced a significant luciferase activity in the TLR-4 transfected cells. Our result can explain in part the immunomodulatory effects produced by DLE both in vivo and in vitro. Work will now focus in the identification of this ligand(s).

This work was supported in part by SIP/IPN. 1Are fellows of COFAA, EDI and SNI.
Use of dialyzable leukocyte extracts (DLE) in patients with severe sepsis (49.35)
Authors: PEREZ-TAPIA SONIA MAYRA , LOPEZ-ISLAS INES , DE LA FUENTE-GRANADA MARISOL , ARRATIA-VALDELAMAR KARINA , JIMENEZ-GALLEGOS NYDIA , RODRIGUEZ-FLORES AZUCENA , ESTRADA-GARCIA IRIS , ESTRADA-PARRA SERGIO ANTONIO
Published: January 2023

Dialyzable leukocyte extracts (DLE) are obtained from freezed-thawed buffy coats and subsequent dialysis ( <12 KDa). They can transfer cellular immune response from an immune donor to a naïve recipient. DLE have been used as immunomodulators for the last 50 years with good results. The sepsis is the main cause of death in the units of noncoronary intensive cares. A controlled clinical study randomized double blind person was made in 24 patients with the diagnosis of severe sepsis and a score of scale of gravity of APACHE II of 20 to 24 points, two groups were created. Group A was administered with one unit of DLE (Transferon) every 24hrs by 7 days, to group Z, was administered with placebo in the same amount and presentation. In both groups the score of APACHE II, IL-1beta, IL-6, IL-8, IL-10, IL-12p70, TNF alpha, PCR, DR/CD14 cells and lymphocyte subpopulations were measured after 1, 2, 3 and 7 days. Results showed that DLE (Transferon) increased DR expression in the CD14+ cells, increased number of Th (CD3+/CD4+) population and decreased the time of stay in UCI
TUMOR-SECRETED VEGF-A DISRUPTS ENDOTHELIAL CELL STIMULATION OF T-CELL, MACROPHAGE AND NK CELL FUNCTIONS (49.5)
Authors: Mulligan Jennifer Konopa , Lathers Deanne M. R. , Young Rita I
Published: January 2023

Our current studies examined tumor-secreted products ability to disrupt endothelial cell stimulation of immune cell functions. Supernatants from EC exposed to tumor-conditioned media (EndoT-sup) contained increased levels of PGE2, IL-6 and VEGF as compared to control supernatants from endothelial cells exposed to media (EndoMedia) or media conditioned by epithelial cells (EndoEpi-sup). Conversely, levels of IL-12 and ET-1 were decreased in EndoT-sup compared to controls. Next we examined NK, T-cell and macrophage functions following exposure to media alone, EndoMedia, EndoEpi-sup or EndoT-sup. NK cell activity measured by TNFα and IFNγ secretion was increased following exposure to EndoMediaand EndoEpi-sup, but reduced following exposure to EndoT-sup. T-cell IFN-γ production was increased following exposure to EndoMedia and EndoEpi-supcompared to media alone. However, T-cell IFNγ and TNFα secretion was reduced following exposure to EndoT-sup. Macrophage function was assessed by microbead uptake showed decreased macrophage phagocytosis following exposure to EndoT-supcompared to controls. Neutralization of tumor secreted VEGF-A restored endothelial cell secretion of IL-12, VEGF and PGE2 to control levels. These studies identify tumor disruption of endothelial cell immune stimulation as a novel mechanism of tumor-induced immune suppression.

Supported by Dept. of Veterans Affairs and NIH.
Reduction of the number and/or suppressive mechanisms of myeloid cells by mammary tumor cells expressing a secreted isoform of MUC1 (50.10)
Authors: Ilkovitch Dan , Herbert Lynn M , Lopez Diana M
Published: January 2023

Expression of the transmembrane isoform of MUC1 (MUC1/TM) in an aggressive murine mammary tumor line, DA-3, results in tumor development leading to metastasis and death of the host, as does the untransfected parental tumor line. However, tumor cells expressing a secreted isoform of MUC1 (MUC1/sec) fail to develop tumors in vivo. The rejection of tumors was found to be immunologically mediated, as initially innate cells and ultimately T cells are required. Previous work to determine which lymphoreticular cells may be involved in the immune rejection revealed that MUC1/sec expressing cells recruited four times the amount of F4/80+ cells, while recruiting significantly lower numbers of GR-1+ cells, relative to MUC1/TM expressing tumor cells. Gr-1+CD11b+ cells known as immature Myeloid Derived Suppressor Cells (MDSC) have been correlated with immune suppression in many models of cancer and chronic infections. MDSC accumulate in response to MUC1/TM expressing tumor cells, but remaine at normal levels in animals exposed to MUC1/sec expressing tumor cells. Futhermore, supernatants from MUC1/sec expressing tumor cells blocked induction of arginase in MDSC, a mechanism of suppression. This block in induction of arginase could also be demonstrated by use of a unique peptide, Immuno Enhancing Peptide, only found in the MUC1/sec protein. NIH, NCI R01CA25583.
LTbR Mediates Cell Contact-Dependent CTL-Directed Anti-Tumor Cytotoxicity (50.11)
Authors: Liu Kebin , Din Najam ud , Browing Darren D , Abrams Scott I , Yang Dafeng
Published: January 2023

Cytotoxic T lymphocytes (CTLs) are thought to use two cell contact-dependent effector mechanisms, perforin- and Fas-mediated cytotoxicity, to suppress tumor growth. Using a CTL adoptive immunotherapy model, we report here that perforin-deficient CTLs (pfp CTLs) exhibited a significant cytotoxicity against Fas-resistant tumors in vivo. We further demonstrated that the anti-tumor activity is directly mediated by the adoptively transferred CTLs. but does not involve CTL-secreted soluble molecules, such as IFNγ and TNFα as lytic agents, since these cytokines exhibited no direct cytotoxicity against these targets. Moreover, it was observed that LTβR, a cell-surface death receptor, is expressed on the tumor cell surface, and LTα, LTβ, and LIGHT, all of which are ligands for LTβR, were either constitutively expressed by or activated in the tumor-specific CTLs and primary CD8+ T cells. Silencing LTβR in these Fas-resistant tumor cells with LTβR-specific shRNA significantly decreased the ability of pfp CTL to mediate tumor rejection in vivo. Taken together, our data suggest that tumor-specific CTL use LTβR-mediated cytotoxicity as a third cell contact-dependent effector mechanism against tumor growth in vivo.
Cooperative downregulation of IFN[gamma]R and Fas is functionally linked to CTL-mediated immunoselection and tumor promotion (50.3)
Authors: Yang Dafeng , Smith Kimberly K , Georgi David , Abrams Scott I , Liu Kebin
Published: January 2023

The host immune system functions as an intrinsic surveillance network in the recognition and destruction of tumor cells. But the immune system, at the same time can “edit” tumor cells and select for tumor variants with reduced immunogenicity through immunoselection. We report here that Fas and IFNγR were coordinately downregulated in the tumor escape variants in vitro. Examination of spontaneously arising mouse primary mammary carcinoma and lung metastases revealed that both Fas and IFNγR protein levels were also dramatically lower in lung metastases than in primary tumors in vivo. Functional disruption of either the Fas-mediated apoptosis pathway, the IFNγ signaling pathway, or both pathways together enhanced the metastatic capability of tumor cells. Gene expression profiling revealed that altered expression of genes involved in immediate IFNγR signaling, the interferon primary response, apoptosis, and tumor colonization are associated with tumor escape. Furthermore, disruption of IFNγR function did not alter tumor cell susceptibility to CTL-mediated cytotoxicity, but is linked to enhanced infiltration of endogenous T cells in the tumor microenvironment and tumor promotion in vivo. These findings suggest that coordinate downregulation of Fas and IFNγR by tumor cells, two key components of cancer immunosurveillance and immunoediting, leads to a more aggressive metastatic phenotype.
The status of IFN-gamma receptor on tumor cells will determine the outcome of tumor relapse or tumor-free survival in FVB mice (50.7)
Authors: Kmieciak Maciej , Kostick Jessica L , Bolesta Elizabeth , Manjili Masoud H
Published: January 2023

We have previously shown that inoculation of the rat neu-overexpressing mouse mammary carcinoma (MMC), derived from FVBN202 mice, into parental FVB mice resulted in the initial tumor rejection and relapse of the neu antigen negative variant (ANV) in a large fraction of FVB mice. Here, we show that status of IFN-gamma receptor alpha in MMC will determine whether this tumor may or may not relapse in FVB mice. We generated MMC transfectants expressing a dominant negative IFN-gamma receptor (dnIFN-gammaRMMC). Animals were then depleted of CD4+ T cells prior to the tumor challenge in order to omit the contribution of the neu-specific antibody and retard the rejection of MMC. Then, we inoculated FVB mice with MMC or dnIFN-gammaRMMC. Interestingly, animals that were depleted of CD4+ T cells at the priming phase of the immune responses failed to reject MMC and showed tumor relapse of the neu negative variant (ANV), but they managed to reject the dnIFN-gammaRMMC with no relapse until 3 months after the challenge. We also showed that status of IFN-gamma receptor alpha in human carcinoma cells SKBR3 will determine IFN-gamma-mediated down-regulation of HER-2/neu in this human carcinoma line. Treatment of tumor cells with IL-6 or TNF-alpha in order to increase IFN-gamma receptor expression rendered tumors more sensitive to IFN-gamma-mediated downregulation of HER-2/neu. Together, our data suggest that determination of the status of IFN-gamma receptor alpha in tumor lesions of HER-2/neu positive breast cancer patients may be a prognostic factor for relapse of their metastatic tumors.
Role of TLR2 and dectin-1 in the host response to Penicillium marneffei (51.10)
Authors: Nakamura Kiwamu , Miyazato Akiko , Saijo Shinobu , Iwakura Yoichiro , Kaku Mitsuo , Kawakami Kazuyoshi
Published: January 2023

Pathogen-associated molecular patterns (PAMP) are recognized by Toll-like receptors (TLR) and C-type lectin receptors including Dectin-1. In the current study, we examined the role of TLR2 and 4 and dectin-1, a β-glucan receptor, in the host response to Penicillium marneffei, an opportunistic fungal pathogen in AIDS patients. IL-12p40 production by bone marrow-derived dendritic cells (BMDCs) stimulated with yeast cells of this fungal pathogens was significantly reduced in TLR2KO and MyD88KO mice, but not in TLR4KO and C3H/HeJ mice, compared to that in control mice. Such production was significantly lower in dectin-1KO mice than wild-type mice. In further experiments, HEK293 cells transfected with TLR2 and/or dectin-1 genes were stimulated with P. marneffei for a NFκB promoter assay. Compared to the cells transfected with control vector, significantly higher activity of NFκB was detected in TLR2-transfectant, but this response was not further enhanced by co-transfection with dectin-1. Taken together, these results demonstrated that TLR2 and dectin-1 played an important role in the recognition of P. marneffei by BMDCs.
Delayed production of TNF-α in neonatal mice in response to Pneumocystis carinii infection corresponds to increased TGF-β levels in the lungs (51.11)
Authors: Rogosky Amy M , Hollifield Melissa , Garvy Beth A
Published: January 2023

Neonatal mice challenged with Pneumocystis carinii (PC) exhibit a three week delay in clearance as compared to adults, corresponding to a delayed CD4+ T cell infiltration into the alveolar spaces. We have published that messenger RNA levels of transforming growth factor (TGF)-β and interleukin (IL)-10 are constitutively expressed in neonatal lungs, suggesting that the lung environment is not adequate to recruit circulating lymphocytes. We have since found that pups have increased TGF-β1 protein levels compared to adults until three weeks of age. The infiltration of CD4+ T cells corresponded to decreasing lung TGF-β levels. Moreover, production of TNF–α by CD4+ T cells was also delayed in pups compared to adults. Ongoing experiments will determine whether TGF-β dampens the proinflammatory response until it is developmentally downregulated or if decreased TGF-β levels follow lung infiltration by activated CD4+ T cells.

This research was supported by PHS grant HL062053.
Heat labile secretory proteins released by Naegleria fowleri induces apoptotic cell death of Jurkat T cells (51.13)
Authors: Shin Myeong Heon , Shin Ho-Joon , Kim Kyeong Ah , Lee Young Ah
Published: January 2023

Naegleria fowleri, a free-living amoeba of soil, ponds, andfresh water, causes primary amoebic meningoencephalitis in humansand other mammals. Upon invasion in vivo, N. fowleri is capable of destructing or killing host cells by cytolysis. However, it is unknown on the mechanism by which N. fowleri induces host cell death. In this study, we investigated the mechanism of host cell death induced by N. fowleri. Incubation of Jurkat T cells with live trophozoites of N. fowleri resulted in cell death, as measured by LDH release assay or propidium iodide staining. In addition, N. fowleri induced DNA fragmentation in Jurkat cells in a time and dose-dependent manner. N. fowleri caused cleavage of calpain, caspase-3, and cytoskeletal proteins such as vinculin and paxillin. Pretreatment of cells with NADPH oxidase inhibitor DPI inhibited N. foweleri-induced apoptosis, whereas calpain inhibitor calpeptin or pan-caspase inhibitor z-VAD-fmk did not. When we incubated Jurkat cells with N. fowleri in a Transwell chamber (pore size 0.4 μm), comparable level of cell death rate was found compared to control without chamber, suggesting an important role of secretory factors in N. foweleri-induced cell death. Moreover, secretroy components released by N. fowleri induced host cell death, and heat treatment abolished host cell death by secretory components. These results suggest that heat labile proteins secreted by N. fowleri induce apoptotic cell death in Jurkat T cells.
Expression of TREM-1 in Macrophages is TLR2 dependent (51.7)
Authors: Choi Esther S , Buckland Karen , Hogaboam Cory
Published: January 2023

Triggering receptors expressed on myeloid cells (TREM) are a group of cell surface proteins that contribute to inflammation, allergy, bone homeostasis, neurological development and coagulation, among other cellular processes. Specifically, activation of TREM-1 on monocytes has been shown to enhance the synthesis of multiple proinflammatory cytokines, chemokines and cell surface molecules in the presence of Toll-like receptor (TLR) specific pathogen associated molecular patterns (PAMPs). Presently, little is known about the regulatory role of TLRs on TREM-1 expression and function in macrophages. Accordingly, we examined the expression and function of TREM-1 in bone marrow derived macrophages (BMMØ) from non-sensitized and Aspergillus fumigatus sensitized wild type C57BL/6 (WT) and TLR2 deficient mice (TLR2−/−). In naïve WT BMMØ, TREM-1 expression was upregulated by IFNγ. BMMØ from naïve TLR2−/− mice did not express TREM-1 transcripts nor was it altered by cytokine or PAMP activation. In the context of sensitization, WT BMMØ expressed greater TREM-1 levels when compared with naive WT BMMØ. TREM-1 was detected in sensitized TLR2 −/− BMMØ but at much lower levels than sensitized WT BMMØ. Finally, both IFNγ and IL-13 increased expression of TREM-1 sensitized WT and TLR2−/− BMMØ. Thus, TLR2 expression was required for TREM-1 expression by BMMØ. Also, TREM-1 expression is markedly increased in BMMØ derived from allergic mice confirming a role for this receptor in allergic responses. Thus, regulation of TREM-1 expression may be a therapeutic target in allergic airway disease.

HL069865
Functional evidence supporting the predicted role of KIR3DS1 in the host response to HIV: KIR3DS1 is an activating receptor expressed by human Natural Killer cells (52.5)
Authors: Pascal Veronique , Yamada Eriko , Martin Maureen P , Alter Galit , Altfeld Marcus , Carrington Mary , Anderson Stephen K , McVicar Daniel W
Published: January 2023

KIR3DL1 is a highly polymorphic inhibitory Killer cell Immunoglobulin-like (KIR) Receptor gene with at least 23 alleles described, including its activating counterpart KIR3DS1. Recently, the KIR3DS1 allele has been shown to slow progression to AIDS in HLABw4*80I individuals. However, due to the lack of specific anti-KIR3DS1 antibody, expression and function have never been reported. In a study of 64 donors, we demonstrate KIR3DS1 expression on a substantial subset of peripheral Natural Killer (NK) cells through its recognition by the monoclonal antibody Z27. The fidelity of this detection method was confirmed by analysis of KIR3DS1 transfectants and the identification of a novel KIR3DS1 null allele. Furthermore, we show that ligation of KIR3DS1 by Z27 leads to NK cell degranulation and interferon gamma secretion. Finally, we document the persistence of KIR3DS1+ NK cells in HIV1 viremic patients. In conclusion, the high frequency of KIR3DS1 expression, together with its ability to activate NK cells, and its maintenance during HIV-1 viremia support the epidemiological data suggesting a critical role for this receptor in controlling HIV-1 pathogenesis.

This project has been funded in part with Federal funds from the NCI and the NIH, under DHHS #N01-C0-12400.
Human vitamin D3-induced antimicrobial activity against Mycobacterium tuberculosis is dependent on cathelicidin (52.7)
Authors: Liu Philip T , Tang Dominic , Krutzik Stephan R , Modlin Robert L
Published: January 2023

One third of the world’s population is infected with Mycobacterium tuberculosis; however, little is known about human host defense mechanisms against this bacterium. We have previously demonstrated that activation of monocytes through TLR2/1 in the presence of sufficient vitamin D3 prohormone (25D3)-induces expression of the antimicrobial peptide cathelicidin, mediated through the conversion of 25D3 into the active hormone (1,25D3). Furthermore, these conditions lead to a VDR-dependent antimicrobial activity against M. tb.; but the role of cathelicidin is unclear. We hypothesize that cathelicidin plays a critical role in the 1,25D3-induced antimicrobial activity, and to address this we utilized the human monocytic cell line THP-1 and siRNA. First, we establish that stimulation of THP-1s with 1,25D3 results in cathelicidin mRNA and protein as well as antimicrobial activity against M. tb.. Using siRNAs specific for cathelicidin, we efficiently knocked down the expression of 1,25D3 induced cathelicidin at both the mRNA and protein levels, whereas a nonspecific siRNA control had no effect. Finally, 1,25D3-induced antimicrobial activity was completely ablated in the presence of siRNA against cathelicidin. Taken together, these data demonstrate that cathelicidin is critically for important host defense against M. tb., and may represent a novel avenue for TB treatment.
Pyrin levels in human monocytes and monocyte derived macrophages regulate IL-1β processing and release (52.8)
Authors: Seshadri Sudarshan , Duncan Michelle Diane , Hart Judith Mary , Gavrilin Mikhail A , Wewers Mark Damian
Published: January 2023

Macrophages and their precursors, monocytes, are key cells involved in the innate immune response. Although both monocytes and macrophages produce caspase-1, the key enzyme responsible for the processing of proIL-1β; macrophages are limited in their ability to process and release functional IL-1β. In this context, since mutations in the pyrin gene (MEFV) cause the inflammatory disorder familial Mediterranean fever, the protein pyrin is believed to regulate IL-1β. To determine whether variations in pyrin levels explain the difference between monocyte and macrophages in IL-1β processing and release, pyrin was studied in fresh human monocytes and monocyte derived macrophages. While fresh monocytes express pyrin protein and MEFV mRNA, monocyte derived macrophages have significantly less pyrin mRNA and protein. Since pyrin levels correlated with IL-1β processing, we asked whether pyrin may promote IL-1β processing and release. Pyrin induced IL-1β processing and release in a dose dependent fashion in HEK293 cells and pyrin overexpression in THP-1 cells confirmed the effect. Conversely, pyrin siRNA suppressed proIL-1β processing in both THP-1 cells and fresh human monocytes. In summary, pyrin expression and IL-1β processing and release is diminished upon the maturation of monocytes to macrophages. In the setting of endotoxin induced activation of mononuclear phagocytes, pyrin appears to augment IL-1β processing and release.

Supported by grant NIH-NHLBI-HL40871 to MDW
Cloning and characterization of equine Toll-like receptor 9 (52.9)
Authors: Zhang Yuwen , Davis Elizabeth G
Published: January 2023

Innate immunity relies on a series of germline-encoded pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), to detect conserved microbial components. TLRs have recently emerged as key PRRs that detect microbial infection and trigger antimicrobial host defense responses. TLR9 is typically expressed intracellularly in immune cells such as dendritic cells. The molecular ligand for TLR9 is unmethylated bacterial CpG DNA. Following cell activation though TLR9, dendritic cells mature and secrete cytokines that initiate naïve T helper lymphocytes to polarize to a Th1 bias. Th1 directed activity is essential for host control of intracellular infection. We cloned and characterized equine TLR9. The deduced equine TLR9 protein has more than 80% homology to that of human, monkey, bovine, canine, feline, porcine, and sheep based on our sequence analysis. We found equine TLR9 expression in spleen, lymph nodes, and peripheral blood leukocytes based on quantitative real time PCR. Flow cytometric analysis revealed that equine TLR9 was expressed in CD4+ T, CD8+ T, and B lymphocytes, particularly in MHC class II expressing cells. Equine TLR9 expression was enhanced by phytohemagglutinin or CpG DNA stimulation. From these data we suggest that the TLR9 ligand, CpG DNA, is a potential equine vaccine adjuvant signaling an antigen-specific Th1 bias for pronounced host immunity.
Complement receptors 1 and 2 influence immune responses in a B cell receptor-independent manner (53.1)
Authors: Jacobson Amanda , Weis Janis , Weis John
Published: January 2023

The CD21/35 proteins are complement receptors which places them squarely within the realm of controlling and interpreting activation states of the innate immune response. The main function for CD21/35 has been identified as a co-receptor for the B cell receptor complex and mice deficient in the CD21/35 proteins have impaired IgG3 antibody responses to a variety of antigens.

We were interested in better understanding the effect of CD21/35 deficiency on immune responses. Our analysis of gene expression profiles of resting and immunized wild type (WT) and CD21/35 deficient animals indicates the enhanced expression of a wide range of inflammatory mediators from CD11b+ cells in the absence of CD21/35 on B cells. Complement activation plays a key role in the differential gene expression observed in the CD21/35 deficient mouse, as depletion of C3 within the animal returned inflammatory gene expression within the spleen to wild type levels. Finally, C3 depletion prior to immunization favors enhanced antigen-specific IgG3 production in the CD21/35−/− mouse, whereas a negative effect on antibody production is observed in the WT mouse. These data suggest that the overall environment of the CD21/35−/− spleen is quite different from that of the WT animal. These differences may directly impact responses, such as IgG3 production, that have previously been considered solely dependent upon the activity of the CD21/BCR co-receptor.

NIH-AI-24158 to JHW and NIH-AI-32223 to JJW
Impaired responses to pneumococcal polysaccharide in CD21/35-deficient mice are caused by dysregulated CD19 expression (53.14)
Authors: Haas Karen M. , Tedder Thomas F.
Published: January 2023

Humoral responses to T cell-independent type 2 (TI-2) antigens, such as pneumococcal polysaccharide (PPS), are critical for providing rapid protection during infections. Complement receptor 1/2 (CD21/35) expression is required for optimal antibody responses, as CD21/35-deficient (CD21/35−/−) mice mount impaired humoral immune responses to TI-2 antigens. CD21/35 associates with CD19, a potent regulator of B cell Src family kinase activity. Altered CD19 expression has dramatic effects on B cell subset development and functional activity. Importantly, CD21/35 deficiency results in significantly increased CD19 expression. To determine whether altered CD19 expression levels in CD21/35−/− mice affect TI-2 responses, CD21/35−/− mice deficient in CD19 were generated and analyzed for their responsiveness to TI-2 antigens, including type 3 PPS and haptenated-Ficoll. CD21/35−/− mice generated impaired TI-2 antibody responses to each antigen, whereas CD19−/− mice generated normal or augmented responses. Remarkably, CD21/35−/−CD19+/− and CD21/35−/−/CD19−/− mice generated normal or augmented TI-2 antibody responses. This data indicates that impaired responses to TI-2 antigens in CD21/35−/− mice are likely to be caused by elevated CD19 activity due to the absence of CD21/35 negative regulation. Thus, CD21/35 regulates CD19 expression and activity to promote optimal humoral responses to TI-2 antigens.
Identification of a phagocytic complement C3d receptor in rainbow trout (53.15)
Authors: Li Jun , Zhang Yong-An , Sunyer Oriol
Published: January 2023

In higher vertebrates, activation of the complement system leads to cleavage of C3 into C3b which binds covalently to complement-activating particles. The C3d degradation fragment of C3b is thereafter produced upon cleavage by factor I and is known to bind to complement receptor type 2 (CR2). The presence of CR2 on B cells enables the uptake of C3d-antigen complexes and together with the B cell receptor, CR2 plays a synergistic role in the activation of these cells. While CR2 has been characterized in mammals, its presence in non-mammalian species remains to be studied. Here we present evidence for the existence of phagocytic C3d receptors in teleost fish B lymphocytes. To evaluate the presence of C3d receptors in trout B cells, we generated FITC-labeled monomers and tetramers of trout C3d and assessed their binding to trout leukocytes. Binding of C3d to trout B cells could be inhibited both by anti-trout C3 antibodies and by unlabeled C3d. Biotinylated C3d was then used to coat 2μm FITC-conjugated streptavidin beads. Trout B lymphocytes were able phagocytose C3d-beads at a much higher ratio than BSA-beads or beads alone. Internalization of beads was inhibited by soluble C3d. To address further the identity of this receptor, we have recently cloned a trout molecule that shows the highest degree of homology to CR1/2 molecules from mammals. Thus far, our results suggest the existence of a trout C3d receptor that has a role in the phagocytosis of C3d-coated particles. Whether this receptor plays a role in adaptive immunity is currently being investigated in our laboratory.
Hematopoietic stem cells did not have a synchronized defect with age-related thymic involution during murine aging (81.5)
Authors: Su Dong-Ming , Zhu Xike , Gui Jingang
Published: January 2023

Previous reports suggested that aging early T-lymphocyte progenitors (ETPs) derived from bone marrow (BM) hematopoietic stem cells (HSCs) have an intrinsic defect and insufficiency in aged thymus, which triggers age-related thymic involution, but our studies suggest otherwise. We grafted fetal thymus into a kidney capsule of the aged mice, in which aging HSCs could fully generate all thymocyte subpopulations and restore normal thymopoiesis under intact physiological conditions in aged animals. We found that aging ETPs were not deficient initially because both BM HSC and early-stage ETPs—DN1a absolute cell numbers were not significantly reduced although late-stage ETPs—DN1b cells significantly decreased. Moreover, proliferation of the aging ETPs remained unchanged although their apoptotic rate increased. Conversely, provision of sufficient young HSCs/ETPs intravenously or intrathymically failed to restore thymopoiesis in aging host mice. Meanwhile, we demonstrated that young progenitors could seed young and aged thymi without significant difference via intravenous injection. The results exclude insufficient and defective HSCs/ETPs as causes for age-related thymic involution. Our findings indicate that an intrinsic defect in aging T-lymphocyte progenitors is unlikely and HSCs are not synchronal defective with age-related thymic involution during murine aging.

(This work was supported by US NIH grant R21AI064939 to DMS).
INVOLVEMENT OF UROKINASE-TYPE PLASMINOGEN ACTIVATOR RECEPTOR IN HEMATOPOIETIC STEM CELL MOBILIZATION (81.7)
Authors: Montuori Nunzia , Selleri Carmine , Ricci Patrizia , Visconte Valeria , Carriero Maria Vincenza , Rotoli Bruno , Ragno Pia , Rossi Guido
Published: January 2023

Cleaved forms of soluble urokinase-receptor (c-suPAR) have been detected in body fluids from patients affected by various tumors. We reported increased c-suPAR levels in sera of healthy donors during granulocyte colony-stimulating factor (G-CSF)-induced mobilization of CD34+hematopoietic stem cells (HSCs). Then, we investigated the effects of uPAR84–95, a c-suPAR-derived chemotactic peptide, on mobilization of mouse CD34+ hematopoietic stem/progenitor cells (HSCs/HPCs). We first demonstrated that uPAR84–95 stimulated in vitro dose-dependent migration of mouse CD34+ M1 leukemia cells and inactivated CXCR4, the chemokine receptor primarily responsible for HSC retention in bone marrow (BM). uPAR84–95 intraperitoneal administration induced rapid leukocytosis, which was associated with an increase in peripheral blood CD34+ HSCs/HPCs. In vitro colony assays confirmed that uPAR84–95 mobilized hematopoietic progenitors, showing an absolute increase in circulating colony-forming cells. uPAR84–95 mobilizing activity was comparable to that of G-CSF; however, neither synergistic nor additive effect was observed combining the two molecules. These findings demonstrate for the first time in vivo biological effects of c-suPAR. Its capability to mobilize HSCs suggests potential clinical applications in HSC transplantation.
Bystander immuno-modulatory effects of human neural stem cell line, HB1.F3, on lymphocyte proliferation induced by mitogens and alloantigens (81.9)
Authors: kim su-young , Kim Jung Sik , Park Chung Gyu
Published: January 2023

A stable clonal cell line of human neural stem cells (NSCs), HB1.F3, has been shown therapeutic effect on acute stroke or intracerebral haemorrhage by the mechanisms of cell replacement and several bystander effects. To investigate the bystander immunomodulatory functions of HB1.F3 cells, we examined immunosuppressive effects of HB1.F3 on T cell effector functions in vitro. We found that heat-labile soluble factors from hNSCs culture supernatant are attributed to the inhibition of peripheral blood lymphocytes (PBLs) proliferation induced by mixed lymphocyte reaction (MLR) or phorbol myristate acetate (PMA)/ionomycin. In contrast to inhibitory effect of HB1.F3, they promoted cytokine secretion from PBLs including IFN-r, TNF-a, IL-10, IL-6 during mixed lymphocyte reaction by soluble factor dependant-manner. HB1.F3 cell line expressed TGF-beta and Trail mRNA, not FasL. we confirmed that the apoptosis induction of PBLs by HB1.F3 is one of the mechanisms of T cell proliferation inhibition during MLRs. In summary, HB1.F3 cells could suppress the T cell proliferation by induction of apoptosis and promote both inflammatory and immune suppressive cytokines. This study suggested that therapeutically effective hNSCs cell line on several stroke models might be due to the bystander immunomodulatory functions by the secretion of immunosuppressive soluble factors.
Growth Factor Independence 1b (Gfi1b) is an E2A Target Gene that Modulates Gata3 in T Cell Lymphomas. (82.3)
Authors: Xu Wei , Kee Barbara L.
Published: January 2023

T cell development depends on the activity of multiple transcription factors, including E2A and Gata3. E2A proteins are required for normal T lymphopoiesis and to prevent T lymphocyte progenitor transformation. Ectopic expression of E2A proteins in E2A-deficient lymphomas results in growth arrest and apoptosis indicating that these cells remain responsive to the targets of E2A. We identify the transcriptional repressor Growth factor independence 1b (Gfi1b) as a target of E2A that promotes growth arrest and apoptosis in lymphomas. Gfi1b expression in primary T lymphocyte progenitors is dependent on E2A and excess Gfi1b prevents the outgrowth of T lymphocyte progenitors in vitro. We also find that Gfi1b represses expression of Gata3, a transcription factor whose appropriate regulation is required for survival of lymphomas and T lymphocyte progenitors. Ectopic expression of Gata3 in lymphomas promotes expression of Gfi1b indicating that these proteins may function in an auto-regulatory loop that maintains appropriate levels of Gata3. Therefore, we propose that E2A proteins prevent lymphoma cell expansion, at least in part, through regulation of Gfi1b and modulation of Gata3 expression.
The regulation of T cell development and leukemogenesis by Ikaros: mechanism of action through Notch. (82.4)
Authors: Chari Sheila , Kathrein Katie , Winandy Susan
Published: January 2023

The development of T cells from multipotent progenitors into lineage-committed cells is dependent upon instructive signals from the microenvironment and the genetic regulatory networks that transmit these signals. The nuclear factor Ikaros (Ik) is a zinc-finger regulatory protein that is essential for normal lymphocyte development. Ik deficiency in mice results in aberrant lymphocyte development, including abrogation of B cell development, hyperproliferative TCR responses, and T cell leukemogenesis. Similarly, the Notch receptor regulates transcription and is required for normal T cell development. Constitutive activation of the Notch pathway in lymphoid progenitors results in similar phenotypes as some of those witnessed in Ik null mice. The collaboration between reduced Ik activity and increased Notch activation in promoting leukemogenesis has been extensively documented for the T cell lineage in humans and mice. However, the molecular mechanisms of this cooperation, as well as the implications of this partnership for thymocyte development and leukemic transformation remain unexplored. In this study, we have assessed the levels at which Notch deregulation occurs in Ik deficient leukemia cells and premalignant thymocytes in terms of Notch cleavage and its target gene expression. We have also examined the extent of Notch activation in the absence of Ik in vitro and ex vivo.
IL-17A-Producing Tn cells escape thymic selection and regulate granulopoiesis in Rag-1−/− mice. (83.16)
Authors: Smith Emily , Stark Matthew A , Zarbock Alexander , Bender Tim , Ley Klaus
Published: January 2023

Interleukin- (IL-) 23 and IL-17A play pivotal roles in the regulation of granulopoiesis in mice via a regulatory neutrostat feedback loop. Dendritic cells (DC) and macrophages release IL-23 in response to microbial products. IL-23 enhances the release of IL-17A from three Tn cell subsets, CD4−CD8−αβlow, CD4+CD8−αβ+ and γδ+. IL-17A levels correlate with neutrophil (PMN) and G-CSF levels in the blood. The feedback loop is closed by the trafficking of PMN’s into the tissues, their apoptosis and engulfment by resident DC, which curbs secretion of IL-23. As most granulopoiesis is controlled by T cells, athymic (nude) mice are neutropenic. However, Rag-1−/− mice have normal PMN numbers and if crossed onto a neutrophillic mouse background, remain neutrophilic, suggesting that immature T cells also regulate granulopoiesis. Here, we show that WT and Rag-1−/− mouse splenocytes secrete IL-17A without TCR cross-linking, and Tn cells are found in the thymus, spleen and MLN of both mouse models. Characterization of these cells places them into the DN1e category of prothymocytes which do not express classical lineage markers and are CD25−, CD117−, CD24− and CD44+. Adoptive transfer of Rag-1−/− thymocytes into nude mice rescued their neutropenia, with 99.8% of PMN’s being recipient derived. Thus, IL-17A-producing thymocytes are able to escape thymic selection and regulate granulopoiesis in the periphery of mice.

Supported by NIH HL 73361