bluuebunny/crossref_metadata_2025 huggingface dataset:
Mast cells play a critical role in host defenses against Yersinia pestis (44.8)
Authors: O’Bryant Deanna M , Nilles Mathew L , Bradley David S
Published: January 2023

There is increasing evidence that mast cells play a role in host defenses, both via an IgE-FcεR mediated, allergy-like, response, as well as an IgG-FcγR mediated response. We provide evidence here that the inhibition of mast cell degranulation greatly enhances susceptibility of HLA-DQ8αβ transgenic (tg) mice to the plague. DQ8 tg mice are significantly more resistant to Yersinia pestis infection compared to out bred Swiss Webster mice, LD50 of >104 and 20 CFUs respectively. The magnitude of the resistance observed in DQ8 tg mice allowed us to examine the contribution of various immunological components during Y. pestis infection. To assess the role of mast cells, DQ8 tg mice were treated with cromolyn, a specific inhibitor of mast cell degranulation, prior to infection with Y. pestis. The enhanced resistance observed in DQ8 tg mice was completely eliminated with cromolyn treatment, suggesting an important role for mast cells in the host response of DQ8 tg mice to Y. pestis. We have previously reported that Y. pestis infected DQ8 tg mice develop a strong total IgG response, while infected Swiss Webster mice do not have any elevation in total IgG levels. The presence of elevated IgG levels may trigger the mast cell response in the DQ8 tg mice. These findings provide further evidence for a role of mast cells in host defenses and provide a model in which the molecular mechanisms of this interaction may be better understood.
Characterization of CCR5 chemokine receptor gene polymorphisms in Japanese and Thai populations (45.11)
Authors: Liu Huanliang , Nakayama Emi E , Theodorou Ioannis , Nagai Yoshiyuki , Likanonsakul Sirirat , Wasi Chantapong , Debre Patrice , Iwamoto Aikichi , Shioda Tatsuo
Published: January 2023

Variations in the human CCR5 gene may alter the expression and/or function of the protein product, thereby altering chemokine binding/signaling or HIV infection of the cells that normally express CCR5 protein. We performed a systematic survey of genetic variations in the entire CCR5 gene as well as CCR2V64I in 50 Japanese subjects and evaluated the effects of those variations on CCR5 promoter activity. We also analyzed CCR5 promoter and CCR2V64I genotypes in 80 more Japanese and 186 Thais. Two types of non-synonymous substitutions in Japanese CCR5 gene were found. Our results showed several novel characteristics of the CCR2-CCR5 haplotype structure that were not reported from studies on Caucasians and African-Americans. Specifically, we were able to show that the G allele at position -2852 from the CCR5 translational start codon in Japanese and Thais is the representative of the CCR5 promoter haplotype that was reported to be associated with rapid progression to AIDS in HIV-infected individuals. Furthermore, nearly all non-synonymous polymorphisms in Japanese CCR5 gene occurred in haplotypes with elevated promoter activity. We thus hypothesized that there was certain selective pressure favoring low levels of CCR5 expression during human evolution.
Increased arthritis and carditis severity in mice coinfected with Borrelia burgdorferi and Babesia microti (46.24)
Authors: Moro Manuel H , Braga Walter U , Zegarra-Moro Ofelia L , Vinasco Javier , Narayanan Sanjeev , Kuroki Keichi
Published: January 2023

The most commonly recognized tick-borne coinfection in most of the eastern USA is Lyme borreliosis and babesiosis, caused by the spirochete Borrelia burgdorferi and the erythrocytic parasite Babesia microti respectively, accounting for ~80% of coinfections. Studies of clinical outcomes indicate that coinfected patients have more severe symptoms and longer duration of illness than patients with Lyme borreliosis alone. Little is known about the immune related mechanisms during tick-borne coinfections. This study used a murine model to examine whether defined disease conditions differed in severity in mice infected solely with B. burgdorferi and in mice coinfected with B. microti and B. burgdorferi. C3H.HeN and BALB/c mice cohorts were coinfected or singly infected and then monitored experimentally for 1, 7, 10, 14, 21 and 60 days after inoculation. Carditis and arthritis was determined by blinded histopathological evaluation of myocardium and tibiotarsal joints. Cytokine measurements were made on lymph node and spleen supernatants for IFN-γ, interleukin (IL-4), IL-10 and TNF-α. Coinfected C3H and BALB/c mice had a significant increase in carditis severity at day 7. In addition, a significant increase in arthritis severity was observed at day 21 and 60 for BALB/c mice. This clinical observation was correlated with a significant increase in the expression of IFN-γ and TNF-α and with a significant reduction of IL-4 and IL-10.

NIH P20RR016443-04
Application of adjuvant therapy for increased innate resistance against Phase II Coxiella burnetii (47.14)
Authors: Lubick Kirk James , Radke Miranda , Wilson Suzanne , Jutila Mark
Published: January 2023

Coxiella burnetii, the causative agent of Q fever, is an obligate intracellular bacterial parasite of macrophages, is classified as a select agent and can be readily weaponized. To date, there is no approved vaccine for C. burnetti in the U.S. and antibiotic therapy is only effective if given very early following initial infection. Infected macrophages can be induced to kill the bacterium, suggesting that “adjuvant therapy” may be an alternative approach to countering infection. As a first test of this approach, the capacity of TLR2 and TLR4 agonists to activate and induce increased killing of Phase II C. burnetii was tested in vitro and in vivo. TLR agonists induced activation and increased killing in vitro, yet showed minimal activity in vivo. In a concurrent drug discovery effort we screened 2,000 natural compounds on macrophage cells as well as on other innate cells for their potential use in “adjuvant therapy”. These novel activating agents identified in the drug screens induced IL-8 release and upregulation of cathepsin D and reproducibly increased clearance of C. burnetii both in vitro and in vivo. These results show that Phase II C. burnetii is susceptible to adjuvant therapy, but TLR agonists are not consistent in inducing protection. Novel agonists from a screen of a natural product library hold promise as potential therapeutic adjuvants for Q-fever.

Funded by: NIH AI-04018 and DoD W91 13M-04-1-0001.
A New Human Adenoviral Type 4 viral vector system displays highly enhanced immunogenicity that is complement-dependent and correlates with altered adaptive responses (47.24)
Authors: Hartman Zachary , Appledorn Daniel , Mendelson Todd , Lyerly Herbert Kim , Clay Tim , Amalfitano Andrea
Published: January 2023

Human Adenovirus Type 4 is widely known as the responsible agent for epidemic outbreaks of Acute Respiratory Disease in military recruits. Although studied for over 50 years, we still have precious little insight into the epidemiology and high viral morbidity surrounding HAdV-4 infections. To address these issues we developed an attenuated HAdV-4 vector system as a basic tool for viral and immunologic research. Our plasmid-based system allows for the manipulation of the HAdV-4 genome, which we used to produce replication attenuated [E1−,E3−] LacZ+ 1st generation Ad4 vectors. Using these vectors, we found that Ad4 elicits an enhanced immunogenic response (comprised of significantly greater IL-6, MCP-1, RANTES, and G-CSF) in murine macrophages in vitro and upon intravenous injection in C57/BL6 mice in vivo. The enhanced immunogenicity is capsid-dependant and is partially complement-dependent in vivo for certain cytokines, related to a more pronounced thrombocytopenia upon i.v. administration. Surprisingly, our studies demonstrate that the HAdV-4 early immunogenicity correlates with suppressed IgG responses, despite different routes of administration for the vector encoded LacZ transgene. These results demonstrate that capsid specific recognition is of paramount importance to both innate and adaptive immune responses and should be an important consideration in vaccine development.
Derivation of synergistically acting, neutralizing, chimeric human-mouse anti-SEB monoclonal antibodies (47.35)
Authors: Tilahun Mulualem E. , Rajagopalan Govindarajan , Osborne Barbara A. , Goldsby Richard A.
Published: January 2023

Staphylococcal Enterotoxin B (SEB) is a superantigen made by S. aureus. SEB can cause food poisoning and shock and its stability and ease of preparation make it a potential agent of bioterrorism. SEB-mediated activation induces T cell proliferation and secretion of cytokines. Antibodies that block the interaction between SEB and MHC or SEB and TCR, can neutralize the activating effects of SEB and provide protection against its debilitating effects. Human IVIG, a therapy for SEB intoxication is of low potency and sometimes, short supply. To surmount we have derived of mouse monoclonal antibodies that block the binding of SEB to MHC at separate and distinct sites. Combinations of these antibodies synergistically neutralize SEB mediated T cell activation. Genes encoding the VL and VH genes of synergistically neutralizing pairs of cell lines were cloned and grafted onto genes encoding either the constant region of human IgH or Igκ and transfected into SP2/0 cells. Chimeric human-mouse antibodies expressed by the transfectants were purified. Tests of these chimeric antibodies demonstrated that they were neutralizing and in combination with each other, still capable of synergistic neutralization of SEB.

Supported by NIH grant # AI057652
Interferon mimetic as a therapeutic for lethal vaccinia virus infection (47.6)
Authors: Ahmed Chulbul M , Martin James P , Johnson Howard M
Published: January 2023

We have developed small peptide mimetics of gamma interferon (IFNγ) that can bypass the poxvirus virulence factor B8R protein and can inhibit vaccinia virus replication in cell culture where intact IFNγ is ineffective. We demonstrate here that the mouse IFNγ mimetic peptide, IFNγ(95–132) with an attached lipophilic group for cell penetration, protects C57BL/6 mice against overwhelming lethal vaccinia virus infection. Intraperitoneal (IP) injection of mimetic before and at the time of intranasal (106 pfu) or IP (107 pfu) challenge with virus resulted in complete protection at 200 ug mimetic and 40 to 60 percent protection at 5 ug mimetic. Initiation of treatment of mice with IFNγ mimetic up to two days post infection resulted in complete protection against death, while initiation of treatment at six days post infection resulted in 40 percent protection. Administration of mimetic by the oral route also completely protected mice against the intranasal route of a lethal dose of vaccinia virus challenge. The mimetic also possessed adjuvant effects in boosting humoral and cellular immunity to vaccinia virus. The combination of antiviral and adjuvant effects by the IFN mimetic probably plays a role in its potent anti-vaccinia virus properties. These results suggest an effective therapeutic against ongoing, lethal poxvirus infections that taps into innate and adaptive host defenses.

Supported by NIH grant AI 056152 and DoD grant W911NF-05-1-0170, both to HMJ.
Tumor cell lysate and adjuvant B7DC-XAb confers effective protection from two antigenically distinct melanomas (48.15)
Authors: Pavelko Kevin D. , Hanson Michael J. , Pease Larry R.
Published: January 2023

Immunotherapeutic approaches to the treatment of melanoma have been hindered by the emergence of melanoma variants and relapsing tumors that are antigenically distinct and hence not recognized by memory cells. We have identified an antibody that has proven effective for the treatment of a mouse model of melanoma, B16. B7DC-XAb is an antibody that specifically binds to dendritic cells and increases their antigen uptake capabilities and their ability to activate T-cells and generates strong cytotoxic lymphocyte responses. We are using B16 tumor cell lysates along with this antibody as an adjuvant for the generation of robust and rapid CTL responses to B16 melanoma. Treatment of B16 tumor challenged mice with B7DC-XAb leads to a CTL response that recognizes an epitope from tyrosinase related protein-1. Subsequent B16-OVA tumor challenge results in a lack of protection, against this related but antigenically unique tumor. Therefore we have used tumor cell lysates in conjunction with antibody treatment in an attempt to broaden the specificity against the melanoma lines. Therapeutic treatment with B16 tumor cell lysate and B7DC-XAb leads to clearance of a 6 day established B16 tumor. Further, re-challenge 30 days later with B16-OVA is also cleared demonstrating an enhanced memory response, to this antigenically distinct tumor. Further studies will determine which antigens are responsible for initiating this response as well as those that are important for tumor clearance.
Conditional triggering of EphA2 epitope presentation results in the enhanced tumor cell recognition by specific CTL. (48.25)
Authors: Mandic Maja , Kawabe Mayumi , Vasquez Anna-Cecilia , Storkus Walter J
Published: January 2023

The receptor tyrosine kinase EphA2 is over-expressed in numerous carcinomas in association with poor clinical prognosis. While pharmacologic agents that antagonize EphA2-mediated signaling could be contemplated as a therapy, a more preferred result would be to decrease tumor cell EphA2 expression while coordinately enhancing specific immune reactivity to tumor cells. We show that specific mAb triggers the internalization of EphA2, resulting in increased processing of EphA2 protein and presentation of EphA2-derived epitopes in tumor cell HLA class I complexes to CD8+ T cells (detected by IFN-γ production and CD107b translocation). Ab-enhanced CTL recognition is blocked by tumor cell co-treatment with Pseudomonas Exotoxin-A and proteasome inhibitors, but not by chloroquine, suggesting that internalized EphA2 complexes are rapidly translocated into the cytoplasm for delivery of peptides into the class I biosynthetic route. When combined with vaccines designed to augment the frequency of circulating anti-EphA2 CD8+ T cells, this agonist strategy may define an effective therapy for patients harboring EphA2+ cancers.
Anti tumor immunity of cyclophosphamide(CY) mediated by homeostatic proliferation of memory type T cells and reduction of regulatory T cells. (48.29)
Authors: Hong Sohee , Yun Il Hee , Lim Jong Hyung , Park Chung Gyu
Published: January 2023

In addition to its cytotoxic effect, CY has been known to have a certain type of immune modulation effects. In this study, we investigated the modulation effect of high dose CY on anti-tumor immunity of tumor bearing mouse under a tumor-mediated immune suppression status. In murine melanoma tumor model, a single injection of CY(200mg/kg) to tumor bearing mouse delayed the tumor progression and increased the mean survival days by 2fold. The phenotypic analysis by FACS revealed CY caused significant increase of both percentage and absolute number of CD4+and CD8+ T cells in draining LNs on 10 days post injection (DPI). Of note, the proportion of CD4+CD25+Foxp3+ T cell was significantly decreased whereas that of CD4+CD44hi and CD8+CD44hiT cell was increased. Caspase-3 staining of CD44hiT cells on 3 DPI indicated the increase was preceded by the early-phase apoptosis. In vitro functional analysis by ELISPOT revealed the number of IFN-γproducing cells stimulated by ConA was increased to the level comparable to tumor non-bearing mouse. We could not find any significant changes in the percentage of monocyte and NK cells with CY treatment. In conclusion, the enhancement of anti-tumor immunity by CY may be due to the increase of the ratio of CD44himemory type T cells to regulatory T cells, which would be induced by early phase depletion followed by preferential homeostatic proliferation of memory type T cells over regulatory T cells.
Phase I clinical and U.S. regulatory experience with designer T cells in cancer (48.33)
Authors: Junghans Richard Paul
Published: January 2023

The process of bringing any product from the bench to the bedside is complex, and particularly so for gene therapy applications. Yet these are some of the most promising agents for the future treatment of cancer and infectious diseases. Designer T cells are autologous T cells retrovirally-modified to express chimeric immune receptors (CIR) against tumor or viral antigens. In this is described our Phase I clinical trial experience with 1st generation anti-carcinoembryonic antigen (CEA) designer T cells in adenocarcinoma and the regulatory processes and timeline for product introduction into the clinic. Further, the regulatory and clinical aspects are summarized for two additional, more advanced designer T cell applications in gastric cancer and prostate cancer that are slated to enter patient trials early in 2007. This information may be of value to investigators who are interested in the translation of laboratory discoveries to human use.

National Cancer Institute, FDA Orphan Product Development Program, Department of Defence, Prostate Cancer Foundation, American Cancer Society
Elimination of Intracranial Gliomas via Immune Rejection of Subcutaneous Allogeneic Glioma Cell Lysate (48.40)
Authors: Stathopoulos Apostolos , Milbouw G. , Chen T.
Published: January 2023

Allogeneic glioma cell lines, coupled with allogeneic and syngeneic tumor cell lysates, could be used to induce immune mediated rejection of syngeneic glioma cell lines in two strains of rat. Fisher344 and Sprague Dawley (SD) rats were implanted subcutaneously with 9L and C6 glioma cell lines. After tumor formation, rats were injected subcutaneously with a combination of allogeneic tumor cell line and combined allogeneic and syngeneic tumor lysate. As a further study, treated rats, who had decreased tumor size from the above treatment, were tested for immune memory. Same experiments were also performed in the intracranial model. Rats treated with the same methode were able to significantly reduce (Fisher344), or completely reject (SD rats), their tumor. “Cured” rats re-injected with five times the original number of malignant cells and remained tumor free. Analysis of these tumors shows numbers of CD4, CD8, B-lymphocytes, and dendritic cells. In the intracranial model, rats had significantly less intracranial spread of tumor than the control group. Our in vivo experiments demonstrate that established subcutaneous or intracranial gliomas may be treated with a combination of allogeneic cells and tumor lysates (allogeneic and syngeneic tumor cells) to reduce its size or eliminate it, and establish lasting memory.
Allogeneic melanoma vaccine expressing alphaGal epitopes induce anti-tumor immunity to autologous melanoma antigens without signs of toxicity (48.7)
Authors: Rossi Gabriela R , Mautino Mario , Lejukole Henry , Koenigsfeld Marie , Vahanian Nicholas , Link Charles J
Published: January 2023

The hyperacute rejection of a xenotransplant is a potent mechanism of tissue destruction mediated by complement-fixing natural Ab that recognize alphaGal (aGal) epitopes. These epitopes are not present on human cells. Humans develop high titers of naturally acquired Ab against these epitopes. We hypothesized that this mechanism could be used to generate a potent anti-cancer vaccine. In this study we tested this hypothesis by using an allogeneic whole cell cancer vaccine to treat murine melanoma. We used the aGal KO mice (aGT H-2bb) and the aGal negative melanoma cell lines B16 and S91M3. We genetically engineered S91M3 (H-2dd) cells to produce the aGal(+)S91M3 cells. Mice with pre-existing subcutaneous and pulmonary B16 (H-2bb) tumors were efficiently treated with aGal(+)S91M3 melanoma vaccines. Control S91M3 vaccines had no impact on tumor growth. Vaccination with aGal(+)S91M3 induced cytotoxic CD8+ T cells recognizing the “autologous” aGal(-)B16 tumors measured by IFN-gamma release and by adoptive transfer experiments. The presence of allo-antigens did not dominate the induction of immunity to “cryptic” tumor antigens tested in the pre-existing melanoma metastasis model. The effective anti-tumor immunity was not associated with autoimmunity or other signs of toxicity in long-term toxicology studies for breast, lung and melanoma allogeneic vaccines. This study establishes for the first time the safety and efficacy data of allogeneic aGal(+) whole cell vaccines and constituted the basis for the initiation of human clinical trials to treat human cancers currently underway.
Lipid Accumulation and Dendritic Cell Function in Cancer (49.10)
Authors: Herber Donna L. , Kroeger Jodi L. , Makris Melissa , Gabrilovich Dmitry I.
Published: January 2023

Immune cell dysfunction in cancer is an area of intense study, with particular focus on dendritic cell (DC) abnormalities. The mechanism of these abnormalities remains unclear. We examined the potential role of lipid metabolism in defective DC function in cancer. Different mouse tumor models (CT26, MC38, or EL4) were used for in vivo studies. Tumors grew for up to 3w and then spleens were harvested for DC analysis. Phenotypic analysis by flow cytometry included CD11c, CD11b, MHCII, CD40, CD80, and CD86. Lipid levels were determined using Bodipy 493/503. For the analysis of DC function, cells with high and normal lipid content were sorted and used in allogenic mixed leukocyte reaction. For in vitro studies, DCs were harvested from spleen and cultured overnight with a variety of stimulants (LPS, tumor supernatant, GM-CSF, or IL-4). The control level of lipids in DCs was established in naïve tumor-free mice. 30–40% of freshly isolated splenic DCs from tumor-bearing mice had increased levels of intracellular lipid compared to naïve mice, and this accumulation was time dependent. Phenotypically, DCs with increased amounts of lipid had increased levels of MHCII and co-stimulatory molecules. However, despite that increase, DCs with high lipid content had substantially reduced ability to stimulate T cells in allogenic mixed leukocyte reaction than DCs with normal lipid amount. These results indicate a role for lipid accumulation in dendritic cell dysfunction. The data also indicate that the tumor microenvironment is necessary to induce the lipid accumulation and dysfunction.
Role of reactive oxygen species in immune suppression in cancer (49.12)
Authors: Corzo Cesar Alexander , Nagaraj Srinivas , Kusmartsev Sergei , Gabrilovich Dmitry
Published: January 2023

The inability of the host immune system to develop and maintain an effective anti-tumor immune response is one of the main mechanisms of tumor progression. Recent findings suggested that hyper-production of reactive oxygen species (ROS) by myeloid cells in cancer may be one of the major factors responsible for T-cell non-responsiveness. However, the mechanism of ROS accumulation as well as their precise role in immune defects in cancer remains unclear. Previous reports have also stated that myeloid-derived suppressive cells (MDSC) contain increased levels of active STAT3 and we set out to address the relationship, if any, between STAT3 and the observed high ROS production. Our data indicate that MDSC isolated from tumor-bearing mice have a significant increase in the expression of two critical components of NADPH oxidase: gp91phox and p47phox than their wild type counterparts. The NADPH oxidase has long been considered a strong source of ROS in myeloid cells and we suggested that STAT3 may be partially responsible for this up-regulation. Our results show that STAT3 does indeed binds to the promoter region of the p47 gene. Furthermore, in vitro inhibition of STAT3 decreased the expression of both gp91phox and p47phox. In addition, the presence of an active NADPH complex was critically important for MDSC-mediated immune suppression. Tumor-bearing mice with defective NADPH oxidase had significantly lower presence of MDSC than their wild-type counterparts. MDSC from gp91phox KO mice had significantly lower suppressive activity against antigen-specific T cells than MDSC isolated from wild-type tumor-bearing mice. Arginase activity and nitric oxide production, factors implicated to mediate MDSC-mediated immune suppression, were also decreased in gp91phox KO mice. These findings may help to clarify the role of ROS in cancer and to develop new effective cancer treatment.
The effects of vitamin C on the interaction between COX-2 expression and IGF-I axis in the human melanoma cell line, SK-MEL-2 (49.27)
Authors: Lee Seung Koo , Kang Jae Seung , Kim Jee Eun , Jung Da Jung , Hahm Eunsil , Bae Seyeon , Hwang Young Il , Shin Dong Hoon , Lee Wang Jae
Published: January 2023

Overexpression of cyclooxygenase-2 (COX-2) frequently occurs in several malignancies and the type I insulin-like growth factor receptor (IGF-IR) is important for growth, transformation, proliferation and protection from apoptosis. However the interaction between COX-2 expression and IGF-I axis in human melanoma remains obscure. Although inhibition of COX-2 could reduce tumor volumes in various murine models, selective COX-2 inhibitors could increase cardiovascular risk. Therefore, we studied the effects of vitamin C, which is known as the potent inhibitor of proliferation of human melanoma cell line, SK-MEL-2. We confirmed that the optimal concentration of vitamin C for the inhibition of proliferation without inducing apoptosis is 1.0 mM. ELISA for IGF-I and IGF-II conducted after treatment with NS-398 and vitamin C revealed that the production of IGF-II was decreased by COX-2 inhibition. RT-PCR for IGF-IR after COX-2 inhibition using NS-398 or COX-2 siRNA showed the inhibition of COX-2 expression led to dramatic down-regulation of IGF-IR expression which is restored by adding exogenous PGE2, which is produced by the enzymatic action of COX-2. Next we observed that both COX-2 and IGF-IR expression were significantly decreased after treatment of 1.0 mM vitamin C. Interestingly exogenously treated IGF-II induced IGF-IR expression and exogenous PGE2 treatment restored IGF-IR expression which is down-regulated by vitamin C. These findings suggest that vitamin C could modulate IGF-II production and then down-regulate the expression of IGF-IR by the inhibition of COX-2 expression, followed by suppression of the proliferation of human melanoma cell line, SK-MEL-2.
Analysis of lymphocyte subpopulations stimulated in vitro with human dialyzable leukocyte extracts (DLE) (49.33)
Authors: GRANADA MARISOL DE LA FUENTE , ESTRADA-PARRA IRIS CITLALLI , CHACON-SALINAS ROMMEL , ESTRADA-PARRA SERGIO ANTONIO , PEREZ-TAPIA SONIA MAYRA
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. Until now little research has been done regarding their mechanism of action. The aim of this work was to analyze, using flow cytometry, if in vitro stimulation of PBMC with a range of DLE concentrations (50 microg, 50 ng, 50 pg, 50fg and 50ag) induced proliferation or activation of different lymphocyte subpopulations. Peripheral blood mononuclear cells (PBMC) were purified using a density gradient and viability was assessed using trypan blue exclusion stain. CFSE stain was performed to measure proliferation of CD3+/CD4+ (Th), CD3+/CD8+ (Tc), CD19+ (B) and CD56+ (NK) cells after 24, 48 and 72h. CD69 expression was measured on the same cell subpopulations after 3, 6, 24 and 48 h. Controls included PMA+ionomicin and non stimulated cells. Results showed that DLE increased CD69 expression only in the Th (CD3+/CD4+) population (50 microg, 50 ng, 50 pg). This subpopulation also proliferate more in comparison with Tc, B or NK cells. Next experiments will analyze if these cells that activated and proliferate upon DLE stimulation express other markes, such as CD25 and Foxp3. Our results may explain in part the immunomodulatory effects that these extracts have.

This work was supported in part by SIP/IPN. 1Are fellows of COFAA, EDI and SNI.
Ligation of CD40 expressed constitutively on breast cancer cell line induces increased TGF-β production (49.8)
Authors: Jung Da Jung , Kang Jae Seung , Kim Jee Eun , Hahm Eunsil , Lee Seung Koo , Bae Seyeon , Hwang Young-Il , Shin Dong Hoon , Lee Wang Jae
Published: January 2023

CD40 has been shown to play a critical role in normal B-cell development and function. Interestingly, CD40 has also demonstrated to be expressed at high levels on some human cancers, including the breast cancer. However, the function of CD40 expressed on tumor cells is still not fully understood. In this study, therefore, the function of CD40 on tumor cells in the aspect of the production of Transforming Growth Factor-beta (TGF-β) was investigated after CD40 stimulation. First, high expression of CD40 on the human breast cancer cell line, MDA-MB231 was observed. The tumor cells were incubated for 24 hr in the presence of agonistic anti-CD40 antibodies or sCD40 ligands, the productions of TGF-β were dramatically increased in each cases. Human T cells purified from peripheral blood were stimulated with PHA and ionomycin. And then cells were cultured with MDA-MB231 for 24 hr. As a result, significantly increased production of TGF-β was observed. Taken together, TGF-β production by breast cancer cells increased via the ligation of CD40 on their surface with CD40L on the surface of T cells. Thus, considering that TGF-β serves dual roles not only in promoting tumor progression and metastasis, but also in immune suppression, these results give an important clue for understanding the mechanism of the immune suppression by tumor cells.
Differential antitumor activity between IFN-lambda and IL-21 (50.18)
Authors: Sato Atsuko , Ohtsuki Mamitaro , Kobayashi Eiji , Murakami Takashi
Published: January 2023

Interferon (IFN)-lambdas are the latest members of the class II cytokine family and exhibit antiviral activity. Murine B16 melanoma and Colon26 cancer cells were transduced with mouse IFN-lambda to determine whether IFN-lambda possesses antitumor activity. Over-expression of IFN-lambda induced Fas, promoted significantly caspase-3/7 activity, and increased p21Waf1/Cip1 and dephosphorylated Rb (Ser780) in B16 cells in vitro. IFN-lambda expression in tumor cell lines markedly inhibited subcutaneous and metastatic tumor formation in vivo. Moreover, an antibody-mediated immune cell depletion assay showed that natural killer (NK) cells were critical to IFN-lambda-mediated tumor growth inhibition. Hydrodynamic injection of IFN-lambda cDNA successfully targeted liver metastatic foci of Colon26 cells, and moderately decreased the mortality of mice with tumors. IFN-lambda over-expression in the liver increased NK cells (2.4 fold) and enhanced their tumor killing activity. However, the antitumor activity of IFN-lambda was weaker than that of IL-21, which also promotes NK-cell proliferation and stimulation. Thus, although IFN-lambda induced both tumor apoptosis and NK cell-mediated immunological tumor destruction, the antitumor effect of IFN-lambda was smaller than that of IL-21.
In vivo analysis of early events leading to T helper cell differentiation (51.15)
Authors: Rozo Cristina T , Liu Qian , Gause William
Published: January 2023

CD4+ T cells are able to respond to various pathogens by differentiating into highly polarized T helper effector (Th) cells that regulate distinct immune responses. Bacteria and viruses can elicit a Th1 response and most helminth parasites can induce a Th2 response. Although, dendritic cells (DCs) are the most potent activators of naïve T cells in vitro, it remains unclear which cell population is most important under in vivo conditions to cause the polarization into effector groups. Naïve CD4+ T cells reside in T cell areas of secondary lymphoid organs and DC’s are the predominant MHC-II expressing cells in the T cell areas. To examine in vivo what antigen presenting cells (APCs) and accessory cells are associated with early stages of the Th2 response C57BL/6 mice received a subcutaneous ear injection in the context of an infectious helminth infection or Th1 inducing agent either in the presence or absence of Ealpha peptide. Local draining lymph nodes were harvested and flow cytometry in conjunction with immunohistochemical techniques were used to visualize antigen presentation using Y-Ae antibody directed towards MHC II complexed with I-E alpha chain and to identify the cell populations participating in the development of the Th2 versus the Th1 immune response. We observed that under Th2 conditions, there was an increase in DX5+ basophils and CD11c+ APCs as early as four hours after immunization.
Neutrophils play critical role for the type 2 protective responses against nematode parasite N. brasiliensis (51.5)
Authors: Liu Zhugong , Pesce John T. , Hamed Hossein , Alem Farhang , Lin Hongxia , Chaussabel Damien , Liu Qian , Urban Joseph F. , Gause William C.
Published: January 2023

Nippostrongylus brasiliensis (Nb) infection induces a potent Th2 response; however, little is known about the innate host-pathogen interactions that lead to the protective Th2 immunity. To identify these early events, microarray analysis was performed on draining skin lymph nodes shortly after Nb inoculation. Expression of several chemokines, including CCL2 and CXCR3 ligands, were found increased. We observed a transient and marked increase in Gr-1+ cells in the draining lymph node after Nb inoculation, which was CCL2-dependent. The Gr-1bright population was identified as neutrophils expressing TGFβ and TNFα. We further demonstrated that neutrophils were required for host protection against Nb infection and for the optimal Th2 development. After neutrophil depletion, Nb infection resulted in early IFN-γ expression, high mortality and delayed worm expulsion. Decreased Th2 cytokine expression and elevated serum IgG2a were also observed. In contrast, infection with Nb larvae treated with antibiotics cocktail to eliminate associated bacteria induced a protective Th2 response in neutrophil-depleted hosts. These studies suggest that the primary function of neutrophils in this model was to eliminate bacteria associated with parasite infection. Neutrophils thus play an essential role in depleting Th1-inducing bacteria, thereby promoting optimal development of the host protective Th2 response.
Perforin-2 Expression in Fibroblasts is Upregulated during dsRNA Stimulation. (52.12)
Authors: Ramos Jahir , Shiratsuchi Motoaki , Deyev Vadim V , Podack Eckhard R
Published: January 2023

Perforin-2 (P2), also referred to as Mpeg1, Mpg1, and MPS1 in the literature, was originally reported as a macrophage specific molecule. Previous reports demonstrated that P2 mRNA was exclusively detected in macrophages and macrophage-like cells. This is the first report that P2 is upregulated in murine fibroblasts during dsRNA or interferon exposure.

Cells were transfected via lipofection with Poly I:C, an analog of viral double stranded RNA. In a separate trial the culture supernatant of Poly I:C- transfected cells was harvested and added to a separate cell culture. Since Poly I:C induces the secretion of interferon, recombinant interferons were used individually and in concert to stimulate a boost in P2 transcription. We also performed promoter assays using luciferase constructs containing various lengths of P2 promoter sequences.

Real time RT_PCR analysis revealed that upon treatment with Poly I:C, P2 mRNA level was increased dramatically. Similar effects were observed following treatment with culture supernatant from Poly I:C-transfected cells. Interferon-alpha, beta, and gamma all induced P2 mRNA upregulation and we found the effect to be synergistic. Our luciferase promoter assay demonstrated that Poly I:C strongly activated an ectopic promoter in exon 1.

Our results indicate that dsRNA stimulation followed by interferon induction increased P2 transcription from an ectopic promoter. We have identified a putative anti-viral responsive gene in murine fibroblasts encoding the membrane anchored P2 protein.
Catalytically inactive cyclooxygenase 2 (COX-2) and absence of PGE2 biosynthesis in murine peritoneal macrophages following in vivo phagocytosis of heat-killed Mycobacterium bovis BCG (52.17)
Authors: Shibata Yoshimi , Shinohara Tsutomu , Tsuji Shoutaro , Henriksen Ruth Ann , Nishiyama Akihito , Myrvik Quentin N , Yamashita Makiko
Published: January 2023

The conclusion that peritoneal macrophages (MØ) isolated from mice given heat-killed BCG (HK-BCG) intraperitoneally do not release PGE2 ex vivo has been established for over 25 years. However, when peritoneal MØ from untreated mice are treated in vitro with HK-BCG, MØ express COX-2, a rate-limiting enzyme for PGE2 biosynthesis, with increased PGE2 release. The present study of C57BL/6 mouse peritoneal MØ treated in vitro or in vivo with HK-BCG was undertaken to determine whether COX-2 is expressed and whether PGE2 release is dependent on its catalytic activity. The results indicate that MØ treated with HK-BCG in vivo expressed COX-2 that was catalytically inactive and localized subcellularly in the cytoplasm but not associated with the nuclear envelope (NE). In contrast, MØ treated in vitro expressed catalytically active COX-2 that was localized in the NE and diffusely in the cytoplasm. In addition, COX-1, constitutively localized in the NE and ER in untreated M , was dissociated from the NE in response to in vivo but not in vitro HK-BCG treatments. These results indicate that COX-1 and COX-2 dissociated from the NE are catalytically inactive, which accounts for the lack of PGE2 production by local MØ activated in vivo with HK-BCG. Our studies suggest that following phagocytosis of intracellular bacteria, local MØ express catalytically inactive COX-2, which may enhance M -mediated innate and Th1 acquired immune responses against intracellular infections sensitive to endogenous PGE2. (NIH RO1 HL71711, DOD DAMD 17-03-1-0004)
Priming of γδ T cells (52.4)
Authors: Hedges Jodi Fern , Buckner Diana , Rask Kerri , Kerns Hannah , Jackiw Larissa , Trunkle Theresa , Pascual David , Dobrinin Erin , Radke Miranda , Holderness Jeff , Jutila Mark
Published: January 2023

We have begun to elucidate the role of γδ T cells in innate immunity. Purified human and bovine γδ T cells display a subtle activation, akin to priming, in response to a wide variety of pathogen associated molecular patterns (PAMPs) such as lipopolysaccharide and peptidoglycan. A primed γδ T cell displays an enhanced response to downstream secondary signals compared to resting cells, but is not overtly activated. The signature response of a primed γδ T cell include increases in Mip1α, GM-CSF, and IL-2Rα gene and protein expression, that renders them more sensitive to IL-2. Priming is distinct from overt activation by known TCR agonists in that upregulation of IFNγ and TNFα, the signature of an activated γδ T cell, are rarely detected. Priming has also been detected in vivo in γδ, but not αβ, T cells derived from the gut mucosa of Salmonella infected calves. In an effort to manipulate γδ T cells to provide an innate protective response, we have identified compounds and herbal products that mimic the priming activation of PAMPs on γδ T cells. Treatment of bovine calves with one of the plant extracts protects against Salmonella-induced enterocolitis. γδ T cell priming may be a critical step in innate immune protection and clearance, especially for pathogens that invade mucosal surfaces, where the majority of γδ T cells are found.
Immune opsonins modulate BLyS/BAFF release in a receptor-specific fashion (53.6)
Authors: Li Xinrui , Su Kaihong , Szalai Alexander J , Zhou Tong , Kimberly Robert P , Edberg Jeffrey C
Published: January 2023

TNF ligand superfamily member 13B (B lymphocyte stimulator (BLyS), B cell activating factor (BAFF)) promotes primary B cell proliferation and immunoglobulin production. It exists in both membrane-bound and soluble forms. Because the soluble form is thought to be the primary biologically active form, we investigated factors that might regulate its cleavage and processing.

In both myeloid cell lines and primary human monocytes, the shedding of membrane BLyS can be regulated by CRP and IgG. Within 10 minutes, both of these opsonins trigger significant shedding of BLyS (up to 52.2±3.0%, p=0.003) by engaging FcγRs. In U937 cells, a primary role of FcγRI is demonstrated both by direct receptor cross-linking (FcγRI 45.69±2.05% verses FcγRIIa 2.11±1.33%) and by the lack of enhanced cleavage when FcγRI and FcγRIIa are co-crosslinked. The shed BLyS is biologically functional since it promotes B cell survival. The generation of a B cell proliferation and survival factor by Fcγ receptor engagement, especially FcγRI, which can also enhance antigen uptake and presentation mediated by ligands of both innate and adaptive immune systems, provides a unique opportunity to facilitate antibody production. These functions suggest that Fcγ receptors may provide a link between immune complex mediated autoimmune diseases and elevations in circulating BLyS in autoimmune disease patients.

Supported by NIH R01AR042476 and R01AR033062.
Control of Germinal Center Reaction against T-Independent Antigens by the Fc(alpha)/(mu)Receptor (53.7)
Authors: Shibuya Akira , Cho Ukiko , Tahara-Hanaoka Satoko , Shibuiya kazuko , Honda Shin-ichiro
Published: January 2023

Germinal center (GC) formation in the secondary lymphoid follicles is induced by immunization with T-independent (TI) as well as T-dependent (TD) antigens. However, structural and functional characteristics of the GC induced by TI antigens have not been elucidated. Here, we show that marginal zone B (MZB) cells and follicular dendritic cells (FDC) preferentially express the Fc receptor for IgM and IgA (Fc(alpha)/(mu)R) as well as the complement receptor CD21/35. Mice deficient in Fc(alpha)/(mu)R showed significantly enhanced GC formation in response to TI antigens, in which both B cells and FDC were involved. Fc(alpha)/(mu)R-deficient mice also showed enhanced TI antigen retention by MZB cells and FDC, CXCL13 expression and affinity maturation of IgG3. Depletion of complements by injection with cobra venom factor completely abrogated this phenotype. These results suggest that Fc(alpha)/(mu)R controls GC reactions and affinity maturation of antibodies against TI antigens by interacting with complement cascades.
Analysis of the in vivo vascular contribution potential of hemato-vascular progenitor cells derived from fetal stages of development (81.1)
Authors: Sánchez Maria José , Garcia Antonio Miguel , Quintero Cristina
Published: January 2023

Hemato-vascular progenitor cells from the adult bone marrow can contribute to blood and vasculature when transplanted into adult irradiated recipient mice. Using this experimental model, high levels of hematopoietic reconstitution can be achieved, while contribution to vasculature is in general very limited. Here we tested if the developmental time of the donor cells and the recipient microenvironment constitute critical factors that determine the extension of contribution to the vasculature.

We have used donor cells from AGM region, fetal liver and adult bone marrow. Cells were obtained from transgenic mice for the SCL-3énh construct that targets vasculature and hematopoietic stem cells, allowing cell lineage tracing in recipient mice. Cells were transferred into the blood stream of adult irradiated and busulfan-treated newborn recipient mice. Using flow cytometry and confocal quantitave microscopy analisys of the liver and kidney vasculature we show that foetal and BM derived progenitor cells contribute similarly to adult vasculature. However, when hemato-vascular progenitor cells are transferered into a new born enviroment, foetal derived cells have a more extended contribution potential to vasculature than adult bone marrow derived cells. This data can have implications for the selection on the donor/receptor ontogenic stage for efficient hemato/vascular transplantation.

Supported by the Spanish Ministry of Education and Science Grant SAF2 07241 and SAF03448/to M.J.S., Junta de Andalucia PAI-CVI 295 supporting grant , Fellowship CONACYT179065 to A.M.G. and fellowhip I3P-CSIC to C.Q.
A Probabilistic Model of Lymphocyte Fate Decisions (81.11)
Authors: Manilay Jennifer O. , Colvin Michael E.
Published: January 2023

New experimental methods for monitoring and manipulating cells down to the single cell level offer vast promise for understanding cell fate decisions in the immune system. An important part of fulfilling this promise will be predictive models of cellular dynamics that can guide experimental interpretation and design. Computational models have been used in stem cell fate studies since Till et al. [PNAS 51:29–36 (1964)] reported a discrete Monte Carlo simulation of stem cell populations that reproduced experimental distributions of proliferative cells in nascent spleen colonies. As part of a combined experimental/computational project to elucidate the regulatory mechanisms underlying T versus B cell development, we have created a probabilistic model of cell differentiation from single common lymphocyte precursors (CLP). In this model, CLP were given probabilities of dividing, death or differentiation that depend on the presence or absence of signaling through the Notch ligand, Delta-1. We used Monte Carlo simulations of 200,000 experiments starting with a single CLP, to predict the resulting distribution of B vs. T cells with different levels of Delta-1. In addition to quantitative predictions to be experimentally tested, this model is a statistical tool for optimal analysis of single cell experiments on differentiating cell populations.

Support: UCM Faculty Startup Funds and DOE (DE-FG02-04ER25625)
Beta-Glucan Induced Mobilization of Hematopoietic Stem Cells Requires Matrix Metalloproteinase-9 (81.8)
Authors: Cramer Daniel , Wagner Stephanie , Li Bing , Liu Jingjing , Hansen Richard , Reca Ryan , Ratajczak Mariusz , Yan Jun
Published: January 2023

Despite advances in stem cell mobilization and techniques, up to 20–25% of patients exhibit poor mobilization and are not able to proceed with auto-transplantation. PGG beta-glucan is a soluble yeast-derived polysaccharide and has been shown previously to induce hematopoietic stem and progenitor cell (HSPC) mobilization. However, the mechanism of action has not been defined. In the current study, we demonstrated that PGG beta-glucan alone was able to mobilize peripheral HSPC at both doses (4.8mg/kg and 9.2mg/kg) after 24 hrs. The combination group (G-CSF/PGG-4.8mg/kg) showed an almost two-fold increase in CFUs compared to the standard of G-CSF alone. Further studies demonstrated that PGG beta-glucan mobilized HSPC via a C or CR3 independent mechanism and did not induce appreciable levels of cytokine secretion. Strikingly, BM cells from PGG beta-glucan mobilized mice secreted abundant matrix metalloproteinase-9 (MMP-9). PGG beta-glucan-induced HSPC mobilization was abrogated in MMP-9 KO mice. Further studies from BM chimeras demonstrated that hematopoietic BM cells and possibly BM endothelial cells stimulated with PGG beta-glucan secreted MMP-9. Taken together, these data suggest that PGG beta-glucan is an agent that enhances HSPC mobilization alone and has a synergistic effect when used in conjunction with G-CSF. This process requires active MMP-9, which results from release of pro-MMP-9 from BM cells.