bluuebunny/crossref_metadata_2025 huggingface dataset:
Small molecule enhancers of peptide binding to MHC class II: Implications for DM function (130.40)
Authors: Call Melissa , Xing Xuechao , Cuny Gregory , Stein Ross , Wucherpfennig Kai
Published: January 2023

MHC II molecules present peptides derived from exogenous protein antigens. Antigens are delivered through the endosomal/lysosomal pathway to the MIIC which contains DM. Nascent MHC II molecules are directed to this same compartment through association with the invariant chain (Ii). During this process Ii is progressively cleaved leaving the placeholder peptide CLIP in the peptide binding groove of the newly translated MHC II molecule. In the MIIC, DM facilitates the exchange of CLIP peptide for high affinity peptides derived from internalized antigens before export of MHC II to the cell surface. Peptides bound to MHC II are generally very stable and can have half lives of days to weeks, while MHC II molecules that have lost peptide rapidly denature, preventing opportunistic binding of unsuitable peptides at the cell surface. These mechanisms greatly hinder peptide immunogenicity as unstructured peptide do not survive to intersect with the MIIC and there is a paucity of empty MHC II receptors on the cell surface to accept exogenously supplied peptides. Through high-throughput screening efforts we have found a number of small molecules that enhance peptide binding to MHC II. One of these small molecules is able to mimic the function of DM at the cell surface and greatly enhances peptide display in vivo. Modeling of putative small molecule interaction sites has led to the discovery of residues that are critical for DM function.
Exposure of murine macrophages to carbon nanoparticles promotes inflammation and inhibits phagocytosis (130.5)
Authors: Suro Raquel , Garza, PhD Kristine
Published: January 2023

Exposure to respirable ultrafine particles (2.5 μM) can adversely affect human health and have been implicated with episodes of increased respiratory diseases such as asthma and allergies; the same is predicted for nanoparticles, which can reach deeper regions of the lung. The objective of this investigation was to examine the effects of carbon nanoparticles on alveolar macrophages. Macrophages (MΦ) function as the first line of defense against invading pathogens and are likely to be amongst the first cells affected by nanoparticulates. We focused on two manufactured nanoparticles: multiwalled carbon nanotube aggregates and black-carbon. The two were tested against MΦ in a chronic contact model. We hypothesized that exposure to nanoparticles would decrease MΦ ability to effectively respond to immunological challenge. Production of interleukin (IL)-12 and nitric oxide (NO), as well as phagocytosis, and MΦ activation were examined in response to lipopolysaccharide (LPS) following a 144 hr exposure to the nanoparticles. Data demonstrated an increase in NO and IL-12 production, a decrease in phagocytosis, and a decrease in the expression levels of activation markers. The data suggests that carbon nanoparticle exposure alters MΦ responses to LPS marked by increased inflammation while potentially limiting clearance and ability to interact with effector T-cells. Thus, physiological exposure to carbon nanoparticles could potentially lead to ineffective pulmonary immunity.
Chronice leptin exposure alters dendritic cell function (130.7)
Authors: Granados Valerie , Ramirez Oscar , Garza Kristine
Published: January 2023

Leptin is a pleiotropic cytokine/hormone that has been shown to play a critical role in immunity, including dendritic cells (DC). In vitro experiments in mice and humans have shown that short term leptin treatment results in enhanced DC function. We hypothesized that chronic exposure, as would occur in vivo, would lead to decreased DC function. Bone marrow (BM) stem cells were thus procured from female C57Bl/6 mice and were treated with leptin during the entire DC differentiation culture period. The chronic leptin-treated BM-DC and control BM-DC (no leptin) were analyzed for phenotype, activation, and phagocytosis. The data varied extensively where leptin treatment resulted in decreased, similar, or enhanced DC function in comparison to controls as determined by expression levels of activation markers, interleukin-12 production, and phagocytosis of fluorescently-labeled E. coli fragments. To evaluate the potential role of female hormones on this outcome, the experiments were repeated with male BM-DC. Interestingly, leptin-treatment consistently decreased DC function relative to controls. The data suggests that constitutive leptin exposure may negatively regulate DC function and is additionally affected by the stage of the estrous cycle in females. The results underscore the complexity of leptin activity on immune cells that is not readily ascertained with short term exposure experiments.
Retrovirally expressed 4-1BBL dramatically increases proliferation and in vivo persistence of adoptively transferred CD19-targeted T cells, promoting complete tumor elimination. (131.16)
Authors: Condomines Maud , Plotkin Jason , Markley John , Gunset Gertrude , Sadelain Michel
Published: January 2023

Human T cells transduced with a chimeric antigen receptor (CAR) composed of an antibody-derived scFv fragment and the CD3ζ chain can recognize and kill tumor cells on binding to the targeted membrane antigen. The co-transduction of a CAR along with CD80 and 4-1BBL induces auto-costimulation enabling high in vivo T cell expansion and tumor rejection in a prostate tumor model. Here we investigated this concept in T cells harboring either a first generation anti-CD19 CAR (19z1) or a second generation CAR (1928z), which additionally comprises the CD28 cytoplasmic domain. We compared the proliferation and anti-tumor activity of 19z1+, 19z1-4-1BBL+, 19z1-CD80+4-1BBL+, 1928z+ and 1928z-4-1BBL+ transduced T cells in a pro B cell leukemia model (NALM-6). Seven days after IV injection into NALM-6 bearing NOD/SCID/IL2Rγnull mice, 1928z-4-1BBL+ CD8 and CD4 T cells accumulated in the bone marrow and the spleen 100 times more than 19z1+ T cells and 10 times more than 1928z+ T cells, corroborating in vitro proliferative responses to repeated antigen exposure. An increased proportion of central memory CD8 T cells was observed in all groups treated with 4-1BBL-expressing cells. Within 15 days, in vivo bioluminescence imaging showed total tumor regression in the 1928z-4-1BBL+ T cell-treated group and partial regression in the other groups. Thus, constitutive 4-1BBL expression drives high proliferation of anti-tumor CD8 T cells, promoting central memory T cell persistence and tumor eradication.
Reconstitution of peripheral CD4+FOXP3+ regulatory T cells in cancer patients receiving adoptive immunotherapy is related to the clinical response to therapy. (131.18)
Authors: Yao Xin , Ahmadzadeh Mojgan , Rosenberg Steven , Robbins Paul
Published: January 2023

The reconstitution of CD4+FOXP3+ T regulatory cells (Tregs) was evaluated in clinical cancer trials that involved the adoptive transfer of in vitro cultured autologous tumor infiltrating lymphocytes (TIL) in conjunction with IL-2. Reconstituting Tregs present in peripheral blood following adoptive transfer were phenotypically and functionally similar to conventional Tregs. Treatment of patients with non-myeloablative (NMA) chemotherapy in conjunction with total body irradiation (TBI) (n=46) prior to adoptive transfer lead to significantly lower levels of circulating Treg cells one to two weeks after cell transfer compared to patients who received NMA alone (n=24), both when evaluated as the percentage of T cells (median 0.98% vs 6.3%, P<.0001) as well as the absolute cell count (2.3/µL vs 45/µL, P<.0001). The intensity of TBI negatively correlated with Treg reconstitution. The frequency and absolute numbers of circulating Tregs one to two weeks post therapy was directly correlated to the number of administered IL-2 doses in the patients receiving NMA alone, but not in the patients receiving NMA plus TBI. Finally, the ratio of Treg cells to total CD3+ T cells was significantly lower in the clinical responders than in non-responders in both NMA (2.4% vs 16%, P<.01) and NMA plus TBI (0.56% vs 4.6%, P<.0001) groups. These findings suggest that strategies that limit Treg reconstitution may enhance clinical responses in cancer patients receiving adoptive immunotherapy.
Provision of CD4+ T cell help preferentially enhances anti-tumor immunity of high avidity T cells (131.19)
Authors: Zhu Ziqiang , Singh Vinod , Bronte Vincenzo , Feigenbaum Lionel , Hurwitz Arthur
Published: January 2023

It has been shown that high avidity tumor-specific CD8+ T cells are superior to low-avidity T cells in anti-tumor activity. However, adoptive transfer of high-avidity CTL populations could still fail as a therapy due to the fact that CTL effector function may be lost following tumor infiltration. To investigate whether provision of CD4+ T cell help will enhance the effectiveness of high avidity CTLs in anti-tumor immunity, we utilized transgenic mice developed in our lab which bear different TCR transgenes specific for a common melanoma differentiation antigen, tyrosinase-related protein 2 (TRP-2(180-188)). T cells from clone 24 Tg mice (TCRhi) show higher avidity than those from clone 37 Tg mice (TCRlo) based on tetramer dissociation and in vitro effector function assays. Here we report that co-activation of CD4+ T cells with TCRhi cells can boost the in vitro function of TCRhi cells, including proliferation, IFN-gamma secretion, Gr-B secretion, and antigen-specific cytotoxicity, but had minimal effects on TCRlo cells. In the treatment of subcutaneous B16 melanoma, we observed that adoptive co-transfer of CD4+ T cells with TCRhi cells (not with TCRlo cells) markedly suppressed tumor growth and induced autoimmune depigmentation at the sites of vaccination and tumor challenge. These findings suggest that provision of CD4+ T cell help preferentially enhances anti-tumor immunity of high-avidity T cells, which may have critical implications for cancer therapy.
Antigen nonspecific memory CD8 T cells expand and gain effector function in response to immunotherapy or to viral infection (131.28)
Authors: Wilkins Danice , Tietze Julia , Alderson Kory , Weiss Jon , Bouchlaka Myriam , Priest Stephen , Taub Dennis , Wiltrout Robert , Longo Dan , Baumgarth Nicole , Blazar Bruce , Redelman Doug , Murphy William
Published: January 2023

Memory CD8 T cells (CD44hi+) have a wide distribution in peripheral blood, secondary lymphoid and non-lymphoid tissues and are hallmarked by their ability to respond rapidly to antigenic re-challenge. We observed that systemic cytokine immunotherapy (IT) results in the marked expansion of memory CD8 T cells due to the preferential expansion of pre-existing memory T cells and not the conversion of naïve (CD44lo+) T cells to an activated phenotype. These CD8 T cells express NKG2D and have cytolytic activity; however, a lack of increased CD25 and PD-1 expression suggests that the expansion and activation are independent of TCR engagement. Studies of CD8 T cells from OT-1 TCR transgenic mice after IT demonstrated the increased lysis of ova-negative tumor targets; moreover, the OT-1 CD8 T cells possessed a memory phenotype and lacked CD25 expression in the absence of ova vaccination. To determine if memory CD8 T cells play a role in pathogenic situations, mice were infected with influenza and tissues were examined for the presence of CD25-NKG2D+ memory CD8 T cells. Interestingly, these cells expanded rapidly in the lungs of infected mice but not peripheral tissues, indicating that memory T cells may play a role in the clearance of pathogen, regardless of antigen specificity. These data suggest that resident tissue memory CD8 T cells may act in reserve as innate effectors and thus bridge the gap between adaptive and innate immunity in cancer immunotherapy and viral infections.
Combinatorial TNF-related Apoptosis-inducing Ligand (TRAIL) plus CpG immunotherapy shows efficacy against metastatic renal cell carcinoma in mice (131.32)
Authors: Norian Lyse , Kresowik Timothy , Kucaba Tamara , Lightfoot Andrew , Rosevear Henry , Schwarz Christopher , Griffith Thomas
Published: January 2023

Metastatic renal cell carcinoma (RCC) is a uniformly fatal disease for which new therapies are urgently needed. Current immunotherapies for RCC have shown some clinical efficacy, but the average survival time for patients remains 6 months. We developed a murine model of metastatic RCC, in which tumor cells transplanted into the kidney spontaneously metastasize to the lungs, then used this model to determine whether immunotherapy administered at the primary tumor site could diminish metastatic tumor outgrowth. Therapy consisted of adenovirus-encoded TRAIL given in combination with CpG 1826. To facilitate quantitation of metastatic tumor spread, we generated a Renca-Luciferase tumor cell line, and analyzed tumor burdens over time via bioluminescent imaging. We found that intra-renal injection of AdTRAIL+ CpG led to a massive influx of dendritic cells and T cells, resulting in diminished primary tumor outgrowth, decreased numbers of lung metastases, and decreased total body tumor burdens. Tumor clearance was dependent upon CD8+ T cells, and required the use of both AdTRAIL and CpG 1826. Thus, a combinatorial immunotherapy that simultaneously increases tumor cell apoptosis via TRAIL, and augments T cell-based antitumor immunity via CpG, when given locally at the site of primary tumor growth, can initiate protective, systemic antitumor immunity against metastatic RCC. Further optimization of such an approach could prove efficacious for patients with inoperable metastatic RCC.
Boosting anti-tumor immunity by combining gp96-Ig vaccination and Ig-mTL1A fusion protein (131.33)
Authors: Khan Samia , Deyev Vadim , Podack Eckhard
Published: January 2023

We previously developed a cell-based vaccine system that secretes heat shock protein gp96 as an Ig fusion protein that efficiently enhances the cross presentation of tumor antigens to CD8+ cytotoxic T lymphocyte (CTL), leading to a tumor antigen-specific clonal expansion. Regulatory T cells (Tregs) in the priming site impose a brake that restricts the full potential of this CTL expansion. In independent studies of TNFR25 and its ligand, TL1A, we found that TNFR25 signaling inhibits the suppressive activity of Tregs and blocks their de novo biogenesis. Combination immunotherapy studies using hamster anti-mouse TNFR25 agonist antibody, 4C12, and vaccine show enhanced tumor rejection in comparison to vaccine only. We have genetically engineered Ig-mTL1A fusion proteins that differ in retention of the cleavage site in the extracellular domain fused to the C-terminus of the mIgG Fc fragment. These fusion proteins are expected to show similar tumor rejection when used in combination therapy with vaccine and, unlike 4C12, are not expected to elicit an antibody response due to prolonged use. The in vitro potencies of the secreted fusion proteins were confirmed by cytotoxicity and binding assays and protein molecular weights were determined by fast protein liquid chromatography (FPLC). We will optimize purification methods to produce the most functional Fc-mTL1A resulting in the strongest TNFR25 signaling, which can be utilized in combination with vaccine for therapeutic benefit.
Polyelectrolyte microcapsules: antigen delivery vehicles for potent induction of CTL responses (131.34)
Authors: Grooten Johan , De Geest Bruno , Naessens Thomas , De Smedt Stefaan , De Koker Stefaan
Published: January 2023

Developing effective cancer vaccines is thought to require the strong activation of cytotoxic T cells (CTLs) to kill transformed cells. Initiation of such CTL responses requires DCs presenting the antigen via the MHCI route, a feature hardly achievable using soluble antigens. Internalization of particulate antigens in contrast has been shown to strongly promote MHCI mediated antigen presentation. In this study, ovalbumin (OVA) was encapsulated in polyelectrolyte microcapsules (PEM), a new class of particles allowing efficient antigen encapsulation under non-denaturing conditions. OVA-loaded PEM strongly promoted MHCI mediated OVA presentation by DCs in vitro and in vivo. Being attractive tools to enhance antigen presentation, PEM however fail to activate DCs, a prerequisite for the initiation of potent CTL responses. To cope with this issue, we coated the surface of cationic PEM with oligonucleotides containing unmethylated CpG motifs, which are strong activators of DCs by triggering TLR9. Co-delivery of CpG and OVA-loaded microcapsules synergistically increased the strength of the CTL response, indicated by a dramatic increase in the number of IFN-γ secreting CTLs and a strongly enhanced killing of OVA peptide-pulsed splenocytes after adoptive transfer. Finally, immunisation with CpG-modified capsules was able to confer protective immunity against a lethal challenge with the highly aggressive OVA-transduced B16 melanoma, clearly demonstrating the potential of this approach.
Interleukin-2 significantly upregulates CD86 expression on T cells during cytokine therapy (131.35)
Authors: Paine Ananta , Kirchner Hartmut , Oelke Mathias , Blasczyk Rainer , Eiz-Vesper Britta
Published: January 2023

Interleukin-2 (IL-2) is one of the most widely used cytokines in therapeutic applications, and its efficacy in the treatment of various cancers and chronic viral infections has been extensively tested. Several studies have focused on the impact of IL-2 on T-cell survival and proliferation. However, very few have focused on IL-2-induced phenotypic changes in T cells, although this is particularly relevant to cytokine therapy. To gain insight into this issue, we analyzed blood samples from patients receiving IL-2 therapy in order to elucidate the phenotype changes occurring in their T cells. A key finding was a marked increase in CD86 expression on CD4+ and CD8+ T cells. Detailed characterizations of the corresponding cytokine-induced CD86 expression profiles on T cells were obtained in subsequent in vitro experiments. We found that the IL-2-induced CD86 molecules are unable to provide costimulatory signals to antigen-specific T cells during activation. Moreover while these CD86 molecules shown to have no binding affinity for CD28 molecules, their binding affinity for the inhibitory molecule CTLA-4 remained intact. Therefore, it is likely that the IL-2-induced CD86 expression on T cells have a modulatory role on IL-2-induced inflammatory response and its related side effects.
Selective targeting of tumor vasculature by cytotoxic T lymphocytes specific for tumor endothelial marker-8 (131.36)
Authors: Perambakam Supriya , Ro Esther , Peace David
Published: January 2023

Tumor endothelial marker 8 (TEM8) is uniquely expressed on tumor vasculature but is absent on normal adult vasculature or somatic tissues, making it an attractive target for immunotherapy. The aim of the current study was to induce human cytotoxic T lymphocytes (CTL) with specificity for TEM8 with the end objective of starving tumors by sustained immunological disruption of their vascular supply. Two HLA-A0201 binding peptide epitopes, TEM8207-215 and TEM8298-306 were identified based on predictive algorithms in silico and T2 binding assays in vitro. Individual CTL cultures were established by stimulation of peripheral blood mononuclear cells with peptides in HLA-A2+ healthy donors. CTL induced with each of these peptides mediated lysis of T2 cells pulsed with the respective TEM8 peptide, but not control HIV-RT476-484 peptide, in chromium release assays. However, only CTL induced with TEM8298-306 peptide exhibited specific lysis of an HLA-A2+ TEM8+ HepG2 tumor cells, indicating that TEM8298-306 is a naturally processed peptide epitope. Further characterization of this CTL demonstrated specific secretion of INF-γ and TNF-α per multiplex cytokine arrays. TEM8298-306 peptide does not share sequence homology with peptides derived from other members of the vWF type-A domain family, notably capillary morphogenesis protein-2 which is widely expressed on normal tissues. These results suggest that TEM8298-306 peptide can be safely used in vaccine formulations for tumor immunotherapy.
CpG-conjugated apoptotic tumor cells elicit potent tumor immunity (131.41)
Authors: Shirota Hidekazu , Klinman Dennis
Published: January 2023

Cancer immunotherapy seeks to induce protective immune responses to tumor-associated antigens (TAA), thereby mediating the eradication of tumor cells. While whole tumor cells represent a comprehensive source of TAA (eliminating the need to identify individual Ags on tumors from different individuals), they tend to be poorly immunogenic. To address this concern, we covalently conjugated immunostimulatory CpG oligodeoxynucleotides (ODN) onto apoptotic tumor cells and examined their ability to induce TAA-specific immune responses. Results show that CpG-conjugated tumor cell vaccines are highly immunogenic. CpG-conjugation enhances the uptake of target cell by DC, triggers the up-regulation of co-stimulatory molecules, and promotes the production of immunomodulatory cytokines (such as IL-12). Vaccination with CpG-conjugated tumor cells stimulates the expansion of tumor-specific CTL that reduce the growth of established tumors. These results suggest that the adjuvant properties of CpG ODN can be harnessed to improve cancer immunotherapy.
T cells with a specific motif in the CDR3-beta region protect mice from CT26 tumor growth (131.46)
Authors: Slansky Jill , Kappler John , Moore Brandon , Dege Carissa , Pennock Nathan , Sprague Jonathan , Franks Alexis , Jordan Kimberly
Published: January 2023

Vaccination with peptide mimics of tumor antigens, or mimotopes, is a potential strategy for antigen-specific immunotherapy of cancer. To determine how to elicit the most effective antigen-specific cytotoxic response against tumors, we have assembled a set of mimotopes of AH1, the immunodominant antigen from the mouse CT26 tumor. Although the AH1 antigen is an ineffective immunogen, vaccination with some of the mimotopes is very successful in protecting mice from tumor growth. The majority of AH1-specific T cells responding to the vaccine or tumor express V-beta 8 gene segments. However, tumor protection by the mimotope vaccines correlates with stimulation of T cells with a more restricted TCR V-beta repertoire. These T cells express receptors that encode a relatively short CDR3-beta motif. This motif consists of a small amino acid, a non-hydrophobic amino acid, a germline-encoded tyrosine, and the J-beta 2.6 gene segment. These results suggest that protective mimotopes enhance antitumor immunity, in part, by increasing the expansion of a subset of functional tumor-specific T cells that naturally respond to the tumor, not a new repertoire of T cells only elicited by the mimotope. We will test the hypothesis that specific T cells with this motif impart increased protection because they have higher affinity for the antigen complex than specific T cells without the motif.
Chicken HSP70 DNA xenogeneic vaccine inhibits tumor growth in a canine cancer model (131.8)
Authors: Yu Wen-Ying , Chuang Tien-Fu , Fischer Laurent , Chu Rea-Min
Published: January 2023

Immunization with xenogeneic DNA is a promising cancer treatment, as it generates immune responses to break tumor tolerance to self-antigens. Heat shock protein 70 (HSP70) is overexpressed in many kinds of tumors and is believed to be heavily involved in tumor progression. This study used a chicken HSP70 (chHSP70) xenogeneic DNA vaccine to elicit immune responses in a beagle canine transmissible venereal tumor (CTVT) model. Two vaccination groups were created: the first (G1) was designed to evaluate the prophylactic efficiency of the chHSP70 DNA vaccine by delivering the vaccine prior to tumor inoculation; the second (G2) was designed to evaluate the treatment efficacy by vaccinating the dogs after tumor inoculation. CTVT dogs received empty vector vaccination and no treatment (CW) were used as controls. Tumor growth was notably inhibited only in the G1 dogs, in which tumors were regressed much earlier than in G2 and CW dogs. The CD4+ subset in tumor infiltrating lymphocytes from G1 dogs was significantly increased than in G2 and CW dogs. The tumor-specific cytotoxicity of PBMC from both G1 and G2 dogs was dramatically enhanced, and ELISpot assay indicated that the canine HSP70 IFN-γ secreting lymphocytes were much more abundant in G1 than in G2 during tumor regression. It was concluded that xenogeneic chHSP70 DNA vaccine successively breaks the tumor tolerance against canine HSP70 and exhibits promising prophylactic activity to inhibit the growth of HSP70-expressing tumor.
An innovative strategy to enhance cross-presentation in dendritic cell-based vaccines (131.9)
Authors: Zhang Tong , Hung Chien-Fu , Wu Tc
Published: January 2023

Immunotherapy has emerged as an important approach for combating cancer. Dendritic cell (DC)-based vaccines are a promising form of immunotherapy because of their ability to induce tumor antigen-specific T cells. Although there are many methods for preparing DC-based vaccines using full-length proteins, cell-penetrating peptides (CPP) have shown success in their ability to deliver whole proteins into DCs and induce antitumor immunity via cross-presentation to CD8+ T cells. This process, however, is limited by the necessity to covalently link the protein of interest to CPP. In our study, we use LAH4, a histidine-rich amphipathic CPP previously used as a DNA transfection reagent, as a novel method of protein delivery that does not require covalent linkage. Our results show that LAH4 mixed with protein could form complexes detectable by immunofluorescence and flow cytometry. We have also shown that LAH4 were capable of complexing with a variety of proteins to bind with different cell types. Furthermore, our data indicate that LAH4 cell binding is partially mediated through surface heparan sulfates. More importantly, DCs incubated with LAH4 mixed with a model antigen, chicken ovalbumin (OVA) protein, were able to significantly activate OVA-specific OT1 T cells compared to OVA protein alone. Thus, we have demonstrated that LAH4 is able to transduce whole protein into cells and has potential to be used as a novel strategy in improving DC cross-presentation in tumor immunotherapy.
Expression and regulation of Toso, a new Fc receptor for IgM, during B cell development (132.15)
Authors: Choi Seung-Chul , Narayanan Sriram , Krzewski Konrad , Borrego Francisco , Coligan John
Published: January 2023

Fc receptors (FcRs) are key regulators of the immune response. A receptor specific for IgM (FcμR) has been recently identified and shown to be predominately expressed on human B cells. FcμR, known previously as TOSO and as Fas apoptosis inhibitory molecule 3 (Faim3), has no role in the inhibition of Fas-mediated apoptosis. We have examined the expression of FcμR in mouse splenocytes by real-time PCR. We found significantly higher levels of FcμR mRNA expression on B cells compared with FcμR mRNA expression on T cells, NK cells, granulocytes, macrophages and monocytes. After stimulation with LPS, anti-CD40 or anti-IgM, FcμR mRNA expression was dramatically down-regulated on splenic B cells. We have also found that bone marrow B cells have lower levels of FcμR mRNA expression than splenic B cells. Furthermore, we analyzed FcμR mRNA expression during B cell development in the bone marrow and we found that immature and mature B cells express high levels of FcμR mRNA compared with pro- and pre- B cells. Currently, we are investigating the role that FcμR plays in B cell development and signaling.
Agonist CD40 antibody mediated stimulation of antigen presenting cells cannot replace CD4+ T help for an effective CD8+ killer response (132.20)
Authors: Ahmed Khawaja , Xiang Jim
Published: January 2023

CD8+ cytotoxic T lymphocytes (CTL) play a central role in the defense against a range of viral, bacterial and parasitic infections. However, the mechanisms by which CD4+ T-cells provide help remain largely elusive. Here, we investigated the concept of replacing CD4+ T-cells requirement for an effective CTL by stimulating antigen presenting cells (APCs) through agonist-CD40 antibody (Ab). C57BL/6 (B6) and MHC class II knockout (KO) mice were intravenously immunized with agonist-CD40 Ab-crosslinked OVA-pulsed DCs (DCova) derived from B6 as well as MHC II KO mice. Consistent with previous work, CD8+ killer response was elicited in MHC class II KO mice, even in the absence of CD4+ T help. Interestingly, DCova from B6 mouse mounted significantly (p<0.05) higher CTL response in B6 mice in comparison to MHC class II KO mice. In contrast, MHC class II KO mouse-derived DCova stimulated lower and roughly similar CTL response both in B6 and MHC class II KO mice. Notably, booster immunization revealed that, although agonist-CD40 Ab endowed DCova (MHC class II KO) the ability to activate CD8+ T killers in the absence of CD4+ T help, but failed to imprint memory programme both in B6 and MHC class II KO mice. Thus, our findings suggest that CD40 ligand (CD40L)-CD40 signaling alone to APCs cannot provide complete help, and besides CD40-CD40L, some other immune components must also be contributing to DC-CD4+ T cell cross-talk for an effective CTL response.
Identification and characterization of highly expressed microRNAs (miRNAs) in memory CD8 T cells (132.21)
Authors: Li Hoi Ming , Weng Nan-ping
Published: January 2023

MicroRNAs are ~22-23 nt single strand RNAs that function in repressing gene expression through inhibiting translation or destabilizing message RNA. Although miRNAs play important roles in lymphocyte development, it is currently unclear if miRNAs regulate differentiation and function of memory T cells. Using a microarray approach, we screened differentially high expressed miRNAs in human memory CD8 T cells and identified five candidate miRNAs that expressed at least two-fold higher in memory than in naïve CD8 T cells. We then analyzed their expression in naïve, central (Tcm) and effector (Tem) memory CD8 T cells by a quantitative RT-PCR method. Only two of them (miR-223 and miR-23a) showed a consistent higher expression in Tcm than in naïve CD8 T cells. Currently, we are analyzing their expression kinetics during activation and aging, and screening their potential target genes.
Altered cellular organization of the splenic marginal zone in aged mice (132.24)
Authors: Birjandi Shirin , Ippolito Jill , Ramadorai Anand , Witte Pamela
Published: January 2023

Splenic marginal zones (MZ) are architecturally organized to generate a rapid response against blood borne antigens entering the spleen. Proper alignment includes MZ B cells, MARCO+ MZ macrophages (MZM), and MOMA1+ metallophilic macrophages positioned around a MAdCAM-1+ cell lined sinus. Our objective was to assess cell specific morphological changes of the MZ in young vs aged mice. We observed gross changes in the spleens of aged mice that reflect the improper alignment of cells in the MZ. Immunocytochemistry and immunofluorescence were used to reveal the positioning of MAdCAM-1+ sinus lining cells and MZM or MOMA1+ macrophages. As reported, in young mice, a continuous single cell layer of MAdCAM-1+ cells enveloped the white pulp, in addition to a cohesive line of MZM and MOMA1+ macrophages closely aligned with MAdCAM-1+ cells. In contrast, the MAdCAM-1+ cells of aged mice were more diffuse and less organized. MOMA1+ macrophages were also discontinuous and highly diffused into the white pulp and B-cell follicles. MAdCAM-1+ cells were surrounded by discontinuous, patchy groups of MZM. The reduction of MZM in aged spleens was confirmed by evaluating the ability of MZM to bind the FITC-labeled Dextran in vivo. By flow cytometry, the reduction in MZM in individual aged mice statistically correlated with reduced frequency of MZB cells. These changes may affect the ability of the MZ to properly respond to and clear pathogens in aged mice. NIH RO1AG013874 and T32AG031780
Internalization of BCR in EU12 µHC+ cells by BCR-Crosslinking cells by BCR-Crosslinking (132.27)
Authors: Marran-Nichol Kathleen , Lui Jing , Zhang Zhixin
Published: January 2023

Engagement of the B cell antigen receptor (BCR) on mature B cells activates signal transduction pathways leading to B cell activation. It has also been shown that crosslinking BCR on murine mature B cells results in partial BCR internalization and limited receptor recycling. The EU12 µHC+ cells are phenotypically and functionally similar to human bone marrow immature B cells. Stimulation of these cells with low concentrations of goat F(ab’)2 anti-IgM antibodies results in complete internalization of the BCR. The internalized BCRs travel to the early and late endosomes, presumably for degradation. Re-expression of the BCR on the cell surface can be detected as early as 2 hours and reach almost normal levels within 6 to 12 hours. However, the reappearance of the BCR, which is completely blocked by Cycloheximide and Brefeldin A treatment respectively, requires de novo protein synthesis and secretion. These results show that the EU12 µHC+ immature B cells do not recycle surface BCR after complete internalization of the BCR.
CD8 T cell response for H60 requires CD4 T cell help when the antigen is expressed as a viral antigen (132.36)
Authors: Kang Bora , Ryu Su Jeong , Choi Eun Young
Published: January 2023

CD4 helper T cell is critical for the generation of primary and memory CD8 T cells specific for non-inflammatory antigens. Minor histocompatibility antigens are representative non-inflammatory cellular antigens that require CD4 help for the response induction, and we have shown that CD8 T cell response for minor histocompatibility antigen H60 is dependent on CD4 help not only for the primary response but also for the secondary response inductions. In this study, we addressed a question whether H60 epitope as a viral antigen would require CD4 help for specific response induction. A recombinant vaccinia virus expressing H60-CD8 epitope and HY-CD4 epitope (rVV-H60HY) was used for immunization of B6 female mice. Immunization with the recombinant virus induced a significant amount of H60-specific CD8 T cell proliferation with a peak frequency around 10~20% of total blood CD8 T cells. The response was CD4 help dependent both after the primary and secondary viral challenges. The CD4 help could be substituted by combined treatment of TLR agonists and anti-CD40 antibody. Our results demonstrate that CD4 help is required for appropriate CD8 T cell responses specific for H60 not only as cellular antigen but also as viral antigen, and the signaling through TLR and CD40 would license antigen presenting cells to appropriately activate antigen-specific CD8 T cells.
Recombinant Rabbit Monoclonal Antibodies to Study Apoptosis and Apoptotic Pathways (132.4)
Authors: Lubkin Margaret , Shallice Matthew , Nyhus Julie , Leong Louis , Aggeler Birte
Published: January 2023

Flow cytometry a tool for studying apoptosis and apoptotic pathways. Using monoclonal antibodies for flow cytometry leads to high specificity for the detection of the target epitope of interest, limiting the use of flow cytometry to available mouse monoclonal antibodies. Here we present high quality recombinant rabbit monoclonal antibodies that do not rely on hybridoma cell lines, but are made with a proprietary recombinant technology to obtain cloned antibodies. These rabbit monoclonals were compared to other available antibodies to demonstrate high specificity and affinity to their targets. We examined lot-to-lot consistency using the same antibody for flow cytometry, western blot and immunocytochemistry. In this study two rabbit monoclonal antibodies involved in apoptotic pathways, Cleaved Caspase-3[Asp175] and p53[pS15] were examined. The p53 antibody is the phosphorylated form of p53 [pS15], which in turn induces p53 Upregulated Modulator of Apoptosis (PUMA), the result being apoptosis through mitochondrial degradation. We performed western blot, and immunocytochemistry studies to show high specificity for both antibodies as well as obtain spatial resolution within the cell.
Lymphopenia precedes the onset of intestinal inflammation in Gimap5 deficient mice (132.6)
Authors: Aksoylar Halil , Barnes Michael , Lampe Kristin , Hoebe Kasper
Published: January 2023

Using N-ethyl-N-nitrosourea (ENU) germline mutagenesis technique, our lab identified a recessive point mutation in a conserved coding region of Gimap5 gene which resulted in severe lymphopenia. The mutation, designated as sphinx, caused total absence of CD8+ T and natural killer cells in the secondary lymphoid organs and caused a progressive loss of peripheral CD4+ T and B cells of homozygous mutant mice with age. Gimap5sph/sph CD4+ T cells adopted a CD44hi CD62Llow effector/memory-like phenotype that is a characteristic of lymphopenia induced proliferation. Additionally, Gimap5sph/sph mice developed intestinal inflammation and wasting disease starting around 8-10 weeks of age which could be reversed by antibiotics treatment and adoptive transfer of wild type splenocytes. Prior to the initiation of colitis, Gimap5sph/sph mice exhibited a reduced total number of regulatory T cells but also a reduced frequency of Foxp3+ cells within the CD4+ T cell population in the spleen and mesenteric lymph nodes. Together, these results establish Gimap5 as a key regulator of lymphocyte development and homeostasis.
Bcl-2 maintains KLRG1low CD127high effector CD8+ and central memory CD8+ T cells by distinct mechanisms (132.9)
Authors: Kurtulus Sema , Tripathi Pulak , Sholl Allyson , Hildeman David
Published: January 2023

After acute infection, most of the effector CD8+ T cells die while some of them survive to become memory cells. Recent work has shown that effector CD8+ T cell subpopulations can be distinguished based on the reciprocal expression of KLRG1 and CD127. During viral infection, KLRG1lo CD127hi effector CD8+ T cells have increased ability to generate memory T cells compared to KLRG1hi CD127lo CD8+ T cells. Here, we assessed the role of Bim and Bcl-2 in controlling the survival of these effector CD8+ T cell subsets. We found that KLRG1lo CD127hi cells had higher levels of Bim and Bcl-2 than KLRG1hi CD127lo effector CD8+ T cells. Using both genetic and pharmacological approaches, we found that survival of most KLRG1lo CD127hi and some KLRG1hi CD127lo effector cells required Bcl-2 to combat the pro-apoptotic activity of Bim. Further, when Bcl-2 was blocked in effector T cells, their development into long-term memory cells was significantly decreased. Similar to subpopulations of effector T cells, we found differential expression of Bcl-2 and Bim in memory T cell subsets. Further, survival of central memory (TCM), but not effector memory (TEM) CD8+ T cells was significantly impaired when Bcl-2 was absent or inhibited. Interestingly, loss of TCM CD8+ T cells could not be rescued by loss of Bim suggesting that Bcl-2 combats molecules other than Bim in CD8+ TCM cells. Combined, these data show the critical role of Bcl-2 and Bim in the control of effector and memory T cell homeostasis.
Native low-density lipoprotein uptake by Macrophage Colony-Stimulating Factor-differentiated human macrophages is mediated by macropinocytosis and micropinocytosis (134.12)
Authors: Anzinger Joshua , Chang Janet , Xu Qing , Buono Chiara , Li Yifu , Leyva Francisco , Park Bum-Chan , Greene Lois , Kruth Howard
Published: January 2023

Human macrophage colony-stimulating factor (M-CSF)-differentiated macrophages—the predominant macrophage phenotype in human atherosclerotic plaques—constitutively take up native low-density lipoprotein (LDL) by fluid-phase pinocytosis, but the pinocytotic pathways mediating LDL uptake by these macrophages have not been thoroughly examined. We identified the kinase inhibitor SU6656 and the Rho GTPase inhibitor toxin B as inhibitors of macrophage fluid-phase pinocytosis of LDL. Assessment of macropinocytosis by time-lapse microscopy revealed that both drugs almost completely inhibited macropinocytosis, although LDL uptake and cholesterol accumulation by macrophages was only partially inhibited (~40%) by these agents. We therefore investigated the role of micropinocytosis in mediating LDL uptake in macrophages and identified bafilomycin A1 as an additional partial inhibitor (~40%) of macrophage LDL uptake that targeted micropinocytosis. When macrophages were incubated with both bafilomycin A1 and SU6656, inhibition of LDL uptake was additive (reaching 80%), showing that these inhibitors target different pathways. Microscopic analysis of fluid-phase uptake pathways in these macrophages confirmed that LDL uptake occurs through both macropinocytosis and micropinocytosis. Our findings show that human M-CSF-differentiated macrophages take up native LDL by macropinocytosis and micropinocytosis, underscoring the importance of both pathways in mediating LDL uptake by these cells.
Identification and characterization of guinea pig interleukin-6 (IL-6) (134.15)
Authors: Brodey Mary , Chen Linda , Cheng Andy , Stollar Neil , Huuskonen Jarkko , Beernink Hans
Published: January 2023

The guinea pig (Cavia porcellus) is an important research model for a number of diseases of the lung, including chronic obstructive pulmonary disease (COPD), tuberculosis, and asthma. Inflammation appears to play an important role in the progression of these various diseases. An important focus in understanding the etiology of these various diseases is the detection and quantification of inflammatory mediators such as cytokines and prostaglandins. Measurements of these inflammatory mediators can also provide a method by which efficacy of potential therapeutics is assessed. A key mediator of inflammation in a number of experimental systems is interleukin-6 (IL-6). The sequence of guinea pig IL-6 has not been published. To enable the development of reagents for the detection and quantification of IL-6 in guinea pig samples, recombinant guinea pig IL-6 was developed through the isolation of mRNA from LPS-stimulated guinea pig splenocytes, amplification of the cDNA, and cloning and expression using appropriate vectors in E. coli. Following refolding and purification, the protein was found to be highly reactive in a B9 mouse hybridoma proliferation assay. The observed ED50 of guinea pig IL-6 was comparable to the observed ED50 of mouse IL-6 (4-8 pg/mL) and human IL-6 (8-20 pg/mL). Sequence analysis revealed that guinea pig IL-6 was approximately 55% homologous with mouse IL-6, and approximately 57% homologous with human IL-6.
DACS-DB: A curation and dissemination model for disease-associated cytokine SNPs (134.19)
Authors: Perumal Narayanan , Bhushan Sushant
Published: January 2023

Cytokines play valuable roles in immune cell growth and lineage specification, and in various disease pathologies. Both low and high throughput studies have implicated the association of cytokine single nucleotide polymorphisms (SNPs) with diseases. A preponderance of such experiments has not shown any causality of an identified SNP to the associated disease. Instead, they have identified statistically significant SNP-disease associations; it is likely that some of these cytokine gene variants may directly or indirectly cause the disease phenotype(s). To fill this knowledge gap and derive study parameters for SNP-disease causality relationships, we have designed and developed the Disease Associated Cytokine SNP Database (DACS-DB). DACS-DB includes data on 456 cytokine genes (derived from the ImmPort database), 10,000 SNPs (from dbSNP), and 330 SNP-associated diseases (from PubMed). We have manually curated functional annotation, heterozygosity and population frequency for the SNPs, and statistical significance of SNP-disease association for diseases. We are currently developing a front end to publish this data on the WWW for dissemination to immunologists and biomedical researchers. The curated data in the DB is to be employed to build a computational tool to predict the disease association potential of an uncharacterized cytokine SNP. This DB can be vital to understand the role of cytokine SNPs as markers in disease and may help identify drug targets for common diseases.
TNF secretion from human mast cells is regulated by mitochondrial dynamics and mitochondrial uncoupling protein 2 (UCP2) (135.11)
Authors: Zhang Bodi , Alysandratos Kostas , Angelidou Asimenia , Kempuraj Duraisamy , Tagen Michael , Vasiadi Magdalini , Asadi Shahrzad , Theoharides Theoharis
Published: January 2023

Tumor necrosis factor (TNF) is involved in T cell-dependent diseases such as psoriasis, and in T cell activation by mast cells, which can both store and de novo synthesize TNF. Yet, regulation of TNF secretion is unknown hampering our understanding of how mast cells regulate allergic, immune and inflammatory processes. Here we show that stimulated human LAD2 leukemic and umbilical cord-derived cultured mast cell (hCBMCs) by SP or IgE/anti-IgE induces degranulation and prestored TNF release, which is accompanied by mitochondrial fission. Images of Confocal microscopy and electronic transmitted microscopy show fragmented mitochondria and relocation from a perinuclear distribution to the cell surface. Moreover, treatment with a mitochondrial fission blocker inhibits degranulation, indicating the necessarily of mitochondria fission in mast cell secretion. Besides mitochondria morphological changes, SP stimulation of LAD2 and hCBMCs increased expression (8 hr) of the mitochondrial uncoupling protein 2 (UCP2), which regulates production of ROS and intracellular calcium, leading to inhibition of subsequent re-stimulation by SP, when is enough for mast cells to recover. Moreover, UCP2 gene expression was reduced, as compared to control normal skin, in affected psoriatic skin, an inflammatory diseases in which mast cells play important role. These findings indicate that mitochondria play a key regulatory role in mast cell TNF secretion and in psoriasis.
Inflammation fails to induce the vitamin D3 1α-hydroxylase. (135.13)
Authors: Ooi Jot Hui , Cantorna Margherita
Published: January 2023

The active form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH) 2D3], is largely produced by a renally expressed enzyme 1α-hydroxylase, which is encoded by the Cyp27B1 gene. Using transgenic mice that have replaced the Cyp27B1 gene with the bacterial lacZ reporter gene (β-galactosidase), we wanted to investigate the conditions that induce extrarenal 1α-hydroxylase expression. Expression of the 1α-hydroxylase in extrarenal tissues including the immune system has been reported. We did not detect β-galactosidase expression in LPS-stimulated BMDM, splenocytes, and peritoneal cells. Moreover, in vivo challenges of LPS, α-galactosylceramide, and Listeria monocytogenes showed no detectable β-galactosidase expression in lung, liver, spleen, and blood. Cyp27B1 expression has been reported in the colon of both DSS-treated and control mice. Cyp27B1 KO mice were more susceptible to DSS colitis than WT mice and showed severe weight loss, inflammation, bleeding, and shrinkage of the colon. DSS treatment of Cyp27B1 KO mice resulted in spotty induction of β-galactosidase in the distal small intestine with a frequency of 3 positives for 22 tested and in the colon with a frequency of 6 positives for 22 tested. No β-galactosidase activity was detected in the intestines of untreated controls. The induction of Cyp27B1 expression was sporadic and not associated with the severity of disease. Our data does not support a role for the induction of Cyp27B1 following inflammation.