bluuebunny/crossref_metadata_2025 huggingface dataset:
New Mouse Models for Immunological Research at The Jackson Laboratory (124.14)
Authors: Merriam Jen , Lutz Cathleen , Rockwood Stephen , Sasner Michael , Donahue Leah
Published: January 2023

The Jackson Laboratory Mouse Repository continues to add to a broad and varied collection of mutant mouse models that are applicable to a variety of research fields and human disease studies. Among the more recent arrivals are a number of humanized strains that are particularly suited for transplantation work, mutations helpful in unraveling the causes of autoimmunity and processes of inflammation, as well as strains that may lead to better understanding of immune cell maturation, recruitment, trafficking and function. Please stop by for an updated list of available strains or visit our website at: http://jaxmice.jax.org/research/immunology/index.html . Donating a strain to the Repository fulfills the NIH’s requirements for sharing of mice. Researchers wishing to have strains considered for inclusion in the Repository are encouraged to submit their strains at: http://www.jax.org/donate-a-mouse .
Antibodies generated against conserved antigens suppress allergic airway disease (125.13)
Authors: Stefanov Emily , Kin Nicolas , Dizon Brian , Kearney John
Published: January 2023

There has been a sharp rise in allergic asthma and asthma-related deaths in the developed world. The hygiene hypothesis proposes that excessively sanitary conditions early in life result in autoimmune and allergic phenomena because of a failure of the immune system to receive proper microbial stimulation during development. Chitin, a biopolymer of N-acetyl glucosamine (GlcNAc), is produced by many allergen-bearing organisms including: fungi, the exoskeleton of insects, crabs, shrimp and parasitic nematodes. We demonstrate that antibodies generated against chitin and other conserved bacterial polysaccharides, induced by neonatal vaccination or passive transfer, are reactive with and dampen the immune response against chitin and Aspergillus fumigatus. In the presence of anti-polysaccharide antibodies, we observed a marked reduction in antigen uptake, cell influx, cellular activation, and cytokine production, resulting in a striking decrease in the severity of allergic airway disease in mice. Our results suggest that antigen exposure, during the neonatal period, from environmental sources, self-antigens, or vaccination, have dramatic effects on the adult B cell repertoire and subsequent antibody responses, modulating the immune response to potential allergens later in life and thus dampening development of allergic airway disease. We propose an adjunct hypothesis that antibodies may contribute to the mechanism of protection proposed by the hygiene hypothesis.
Methylation patterns of COX-2 promoter affect gene transcription and PGE2 levels post-bone marrow transplant (126.23)
Authors: Domingo-Gonzalez Racquel , Huang Steve , Moore Bethany
Published: January 2023

Alveolar macrophages (AM) and polymorphonuclear leukocytes (PMN) are critical for innate immune host defense and the function of these cells is impaired post-syngeneic bone marrow transplant (BMT) due to overproduction of prostaglandin E2 (PGE2). The rate limiting enzyme for PGE2 synthesis is cyclooxygenase-2 (COX-2). This study sought to learn why COX-2 and PGE2 levels were elevated post-HSCT. Our studies show COX-2 transcription is increased in BMT AMs relative to control AMs. This is associated with hypomethylation of the COX-2 gene near the transcription start site and into exon 1. Treatment of primary AMs or MHS cells with 5-aza-2’-deoxycytidine increased COX-2 transcription and PGE2 synthesis. Transfection of MHS cells with in vitro methylated COX-2 promoter-driven constructs decreased luciferase activity relative to transfection of unmethylated constructs. Our results demonstrate COX-2 expression is highly regulated by DNA methylation. Furthermore, hypomethylation of the COX-2 gene occurs post-BMT and this correlates with increased expression of COX-2 and PGE2 in AMs from BMT mice. Thus epigenetic changes in COX-2 likely impair innate immune defense in the lung post-BMT. Our current studies suggest that transforming growth factor β may be one factor which regulates these epigenetic changes post-BMT.
Role of PAMPs and DAMPs in Graft Rejection (126.6)
Authors: Chain Robert , Varghese Linda , Gallucci Stefania
Published: January 2023

Dendritic cells (DCs) induce immunity or tolerance depending on the presence of danger signals. LPS is a danger signal with multiple effects on DCs, besides activation. Unpublished results from our lab show that LPS induces DC death in vitro and in vivo. It has also been reported that DCs treated with LPS during their development remained immature and induced T cell anergy. LPS triggers TLR4, a PRR also stimulated by endogenous danger signals, like HMGB1, released during tissue damage. We used a mouse transplant model to determine the effects of simultaneous exposure to LPS and endogenous danger signals, released during engraftment, on DC functions and graft survival. We used the single minor male-specific H-Y antigen mismatch. We transplanted skin of male C57BL/6 mice onto syngeneic female recipients. We administered 4 i.p. injections of 12.5ug/mouse of LPS or PBS every other day. Control mice rejected male skin graft in 24-34 days, as expected, while mice treated with LPS did not reject the graft until 64 days or later. Studying the DCs migrating out of the graft, we found a sharp decrease of DCs 48 hours post transplant and the DC loss was more severe after LPS. LPS also decreased the number of DCs with high surface expression of MHC class II. These findings suggest that the combination of endogenous danger signals released during engraftment and LPS are either killing the DCs or preventing them from maturing thus allowing for tolerance of the male skin graft.
IFNγ production by donor-reactive memory CD4 T cells is required for CD40-independent induction of pathogenic alloantibody (126.8)
Authors: Gorbacheva Victoria , Ayasoufi Katayoun , Fan Ran , Yu Hong , Valujskikh Anna
Published: January 2023

Donor-reactive memory CD4 T cells are potent inducers of alloantibody (alloAb) even in the absence of the CD40/CD154 pathway. The goal of this study was to determine the cytokine requirements for CD40-independent help by memory CD4 T cells. Using donor-specific TCR transgenic T cells, we found that Th1 and Th17 memory CD4 T cells elicited high titers of alloAb in CD40-/- heart allograft recipients while Th2 cells failed to do so. The resulting anti-donor Abs differed in their specificity and the ability to mediate heart allograft pathology. Compared to Th1 cells, Th17 memory cells induced inferior Ab responses against donor MHC class I molecules. Anti-donor alloAb induced by Th1, but not by Th17 memory cells, caused diffuse C4d deposition in the graft capillaries. Blocking IFNγ in CD40-/- heart allograft recipients containing memory CD4 T cells drastically reduced serum IgG alloAb titers. In contrast, donor-reactive CD8 T cells were efficiently activated under these conditions indicating that the help for CD8 T cells is IFNγ-independent. Our data suggest that IFNγ secreted by donor-reactive memory CD4 T cells not only determines donor-reactive Ab isotypes, but also influences alloAb specificity and can thus affect allograft pathology.
Adenosine A2B receptor signaling in antigen presenting cells suppress anti-tumor adaptive immune responses (127.10)
Authors: Cekic Caglar , Linden Joel
Published: January 2023

Adenosine is generated in response to cellular stress such as hypoxia and inflammation and acts as a “metabokine” to modulate immune responses and prevent tissue damage. The accumulation of high levels of adenosine in tumors activates adenosine A2A and A2B receptors on immune cells to attenuate tumor rejection. Previously, we have shown that A2BR blockade inhibits growth of syngeneic tumors by enhancing innate and adaptive immune responses. However, the cellular targets that mediate inhibition of tumor growth after A2B receptor blockade are unknown. In this study, we tested the activity of innate and adaptive immune cells isolated from bladder or melanoma tumors grown in mice reconstituted with mixed bone marrows from A2BR-/- and A2BR+/+ animals. While A2BR deficiency did not influence the activation of NK cells and T cells, A2BR-/- antigen presenting cells were activated more than A2BR-/- cells. Selective deletion of A2BRs on myeloid cells or CD11c expressing cells significantly delayed the growth of syngeneic melanoma. The results show that antigen-presenting cells are direct cellular targets for tumor growth inhibition after A2BR blockade. The results of this study have important implications for therapeutic use of A2B receptor blockade or deletion for treatment of cancer.
Identifying an IDO-expressing “fibrocyte” subset of myeloid suppressor cells in pediatric cancer patients. (127.28)
Authors: Zhang Hua , Mackall Crystal
Published: January 2023

Human myeloid derived suppressor cells (MDSC) have been described as CD14-CD11b+CD33+HLADR- cells that exert T cell suppression via ROS, ARG1 and iNOS2. Here, we identify a novel myeloid suppressor subset, expanded in the peripheral blood of pediatric cancer patients, which is CD14-CD11b+CD33loHLADR+. Further analysis revealed that these abnormal myeloid cells were CD34+CD11chiIL4Rα+CD16+, and express α smooth muscle actin and collagen I/V on their surface, hallmarks of fibrocytes. The fibrocytes expressed high levels of indoleamine oxidase (IDO), and suppressed T cell proliferation in response to OKT3, which was partially reversed by the addition of 1MT, an inhibitor of IDO. Given the IL4Rα expression, we sought to induce these cells in vitro from normal monocytes using IL-4. Indeed, IL4 alone was sufficient to drive monocytes to differentiate into fibrocytes, which demonstrate a spindle-like shape, adhere to plastic, acquire the CD14-CD34+CD11chiCD123-CD16+collagenI+ phenotype, and express IDO. Fibrocytes have previously been implicated in chronic inflammation, angiogenesis and wound healing. We conclude that they also constitute a novel subset of myeloid derived suppressor cells in humans, and could contribute to tumor progression.
Imbalance of activating and inhibitory receptor expression on effector cells in multiple myeloma patients results in immune suppression (127.35)
Authors: Hsu Andy , Davenport Alex , Smyth Mark , Ritchie David , Neeson Paul
Published: January 2023

Multiple myeloma (MM) is an incurable plasma cell malignancy of the bone marrow (BM). Recent evidence suggests that natural killer (NK) cells are intricately involved in the immunosurveillance of MM and can specifically target malignant plasma cells in the absence of the immunosuppressive BM microenvironment. Immune suppression in MM patients is systemic with total CD4 T cell counts in the peripheral blood significantly lower than healthy donors. We have data showing that the major NK activating receptor NKG2D is significantly reduced in MM patients, whereas inhibitory NKG2A, KIR2DL1 and KIR2DL3 are up-regulated. These inhibitory receptors target HLA-E and C respectively, and dominate over activating signals. Interestingly, we report that NKG2A, KIR2DL1 and KIR2DL3 were also up-regulated on T cells in MM patients. Our subsequent investigations showed that expression of KIR receptors could be induced on CD4 T cells independent of direct cell contact with MM cells. Throughout treatment using an empirical MM drug regimen including an immunomodulatory drug and a corticosteroid, we saw restoration of NKG2D but no change in the expression of inhibitory receptors on NK or T cells in MM patients. Together, this data reveals a novel model of T cell tolerance to MM and highlights to therapeutic potential of KIR blockade to reverse immune suppression.
M2 macrophages express CD1d and are selectively targeted by NKT cells in tumors (127.39)
Authors: Courtney Amy , Liu Daofeng , Wei Jie , Gao Xiuhua , Marinova Ekaterina , Asgharzadeh Shahab , Metelitsa Leonid
Published: January 2023

CD1d-reactive Valpha24-invariant Natural Killer T cells (NKTs) are potentially important for tumor immunity. However, the mechanism by which NKTs mediate antitumor responses against CD1d-negative tumors has remained enigmatic. Recent findings by us and others suggest that instead of attacking tumor cells directly, NKTs may target CD1d-positive tumor-supportive myelomonocytic cells. We have demonstrated that NKTs co-localize with tumor-associated macrophages (TAMs) in primary human neuroblastoma (NB). The phenotypic analysis of TAMs in primary tumors has revealed the existence of both M1- and M2-polarized subsets. However, only the numbers of CD163-positive M2 macrophages correlated with metastatic disease and poor outcome. Importantly, we found that CD1d is selectively expressed on M2, but not on M1 macrophages, both in TAMs isolated from primary NB tissues, and in in vitro polarized macrophages. NKTs mediated CD1d-dependent in vitro cytotoxicity against M2 macrophages or polarized them to an M1 phenotype via soluble mediators. The latter was in part dependent on NKT-derived GM-CSF and IFNγ. Moreover, adoptive transfer of ex-vivo expanded human NKTs resulted in a significant reduction that correlated with anti-metastatic activity. Thus, targeting of tumor-supportive M2 TAMs by NKTs reveals a novel mechanism of tumor immunity that can be exploited for the development of effective immunotherapy of solid tumors.
Interleukin 18 regulated gene, erythroid differentiation regulator 1 acts as metastasis suppressor in murine melanoma. (127.5)
Authors: Jung Min Kyung , Cho Daeho
Published: January 2023

Erythroid differentiation regulator (Erdr1) was first discovered on mouse leukemia cell lines and functions as a stress-related survival factor. This study investigated whether Erdr1 regulates murine melanoma progression, as well as the mechanism involved in Erdr1-regulated metastasis. The expression of Erdr1 is negatively correlated with IL-18 expression which has a pro-cancer effect in melanoma. To study the role of Erdr1 as anti-cancer factor, cell migration and invasion ability were measured. Erdr1 over-expression markedly inhibited the level of cell migration and invasion ability in B16F10 cells, in vitro. Additionally, Erdr1 over-expression significantly suppressed melanoma lung colonization and metastasis, in vivo. In order to indentify the factors involved in Erdr1-reduced metastasis, heat shock protein 90 (HSP90), a well known stress protein and contributor to tumor metastasis, was examined. We found that HSP90 was significantly decreased in Erdr1 over-expressing cells. Functional analysis demonstrated that HSP90 siRNA transfection reduced the migration ability and metastasis of melanoma. In conclusion, Erdr1 shows powerful anti-metastasis effect that leads to ability to reduce the metastatic potential of murine malignant melanoma cells. Erdr1 is a novel anti-metastatic factor and may be a possible therapeutic target for treatment of melanoma.
Lysosomal DNaseII degrades nuclear DNA and prevents self DNA recognition (159.17)
Authors: Lan Yuk Yuen , Londoño Diana , Hacohen Nir
Published: January 2023

The aberrant activation of innate immune sensors by self nucleic acids can foster the development of autoimmunity. For example, the absence of nucleases that degrade self nucleic acids leads to autoimmune diseases in mice and humans. DNaseII is the major lysosomal DNA-degrading nuclease. Its deletion in mice results in the accumulation of self DNA, induction of inflammatory cytokines, and development of chronic polyarthritis that resembles human rheumatoid arthritis. DNaseII is known to degrade extracellular DNA in phagocytes. However, given its ubiquitous expression across cell types, we hypothesized that the nuclease plays a broader role in the clearance of DNA. Supporting this notion, DNA accumulated in both phagocytic and non-phagocytic cells from DNaseII-deficient mice, and could be further elevated by treatment with DNA-damaging agents. Concomitantly, cytokines and other immune genes were upregulated through a Sting-mediated cytosolic nucleic acid sensing pathway. Surprisingly, DNA accumulated within nuclear buds and extranuclear aggregates that often co-localized with autophagic and lysosomal markers. A deficiency in autophagy displayed higher levels of extranuclear DNA and enhanced Sting-dependent innate immune response. The observed nuclear to lysosomal DNA clearance process appears to eliminate discarded nuclear DNA in the steady state and in response to DNA damage, and protect cells and organisms from developing pathological immunity to self DNA.
IL-17-induced Regulator of G-protein Signaling orchestrates germinal center kinetics in the onset of autoimmunity in BXD2 mice (159.24)
Authors: Mountz John , Wang John , New James , Yang PingAr , Wu Qi , Druey Kirk , Hsu Hui-Chen
Published: January 2023

Development of germinal centers (GCs) is a hallmark of BXD2 autoimmunity. We have shown that IL-17+ CD4 cell frequencies are significantly elevated in the spleen of BXD2 mice, leading to the induction of Regulator of G-protein Signaling (RGS) 13 and 16 and decreased sensitivity to CXCL12 and CXCL13. The resulting chemotactic arrest within the GC promotes extensive somatic hypermutation (SHM) mediated by activation-induced cytidine deaminase (AID). Here we explore mechanisms underlying this phenotype using BXD2-Rgs13-/- and BXD2-Rgs16-/- mice. BXD2-Rgs13-/- mice exhibited higher autoantibody titers in early age compared to wild-type (WT) mice though smaller GCs and lower AID levels suggested a lesser extent of SHM and affinity maturation. There was upregulation of IRF4 in B cells of BXD2-Rgs13-/- mice, suggesting these B cells develop into plasma cells more rapidly than WT B cells. At older ages, BXD2-Rgs13-/- mice showed less IgG deposits in the glomeruli, suggesting reduced autoantibody pathogenicity. RGS16 deficiency led to aberrantly localized T cell zones and reduced aggregation of a population of CD86hi marginal zone precursor (MZP) B cells, accompanied by significantly fewer GCs, suggesting a role in localization of MZP in GC light-zone and B-T co-stimulation events. Taken together, these results suggest a unique role for IL-17 in inducing pathogenic GCs and development of autoimmune disease in BXD2 mice through RGS13 and RGS16-dependent pathways.
Early biomarkers of resistance and susceptibility to mycobacterial disease (160.1)
Authors: de Silva Kumudika , Begg Douglas , Plain Karren , Purdie Auriol , Kawaji Satoko , Dhand Navneet , Whittington Richard
Published: January 2023

Mycobacterial diseases such as tuberculosis and paratuberculosis continue to be of serious health and economic concern to humans and livestock globally. Lack of knowledge on the nature of the protective immune response as well as immunological markers of disease susceptibility and latency hamper the development of effective vaccines and other therapies. The use of a well-characterised ruminant infection model enabled us to follow the host response to mycobacterial challenge from the time of exposure until manifestation of clinical disease in a natural host. This study (30 controls and 58 challenged in 2 trials) tracked cellular and humoral responses, and quantity of mycobacterial shedding, for up to 30 months post challenge in sheep. Infection was defined as the presence of viable organisms in tissue sections taken at necropsy. The IFNγ response increased after exposure regardless of disease outcome. Parameters measured could distinguish severely diseased animals from those that had been exposed but were uninfected. Infectious or severely diseased animals were more likely to have a lower IFNγ response early (4 months post exposure) compared to exposed uninfected animals. This latter group were also more likely to have an elevated early IL-10 response. These studies demonstrate the complexity of the immune response to pathogenic mycobacteria and illustrate the fact that measurement of a single immunological parameter is insufficient to determine the outcome of disease.
Cytotoxic T cell epitope in Brucellosis-induced murine spondyloarthropathy shows 100% homology with the host mouse peptide (160.6)
Authors: Adarichev Vyacheslav , Lyons Elizabeth , Magnani Diogo , Forde Toni , Splitter Gary
Published: January 2023

Brucellosis is resulted in spondyloarthropathy (SpA) in almost 50% of human patients, but mechanisms of this association are poorly characterized. To study immune mechanisms of SpA, BALB/c mice were infected with bioluminescent B.melitensis, and bacterial dissemination in spleen, liver and axial skeleton was monitored using bioluminescence and immunohistochemistry (IHC). At the peak of infection, bioluminescence showed accumulation of bacteria in front and hind paws, but the strongest signal was detected in the caudal skeleton reproducing the distribution of vertebrae. IHC confirmed intracellular bacteria in spleen, liver, and in the subchondral bone marrow of vertebrae and in peripheral joints without inflammatory cells infiltration. At 26 weeks post infection, bacteria were cleared from the body, but mice developed ankle joint arthritis and spondylolisthesis. We found that immunodominant Brucella-derived peptide from methionine sulfoxide reductase is presented by murine macrophages in context of MHC class I molecule to cytotoxic T cells (CTL). Homologous mouse/host protein carries a sequence that is 100% identical to Brucella epitope. In silico prediction analysis indicates that Brucella epitope binds to human HLA-B*2705 and B*2702 class I molecules, that are strongly associated with human SpA. Our data provide support for brucellosis-induced SpA as a reactive arthritis-type disease with a potential involvement of CTL in the disease pathogenesis.
IL-33 directly supports the proliferation of suppressive, naturally-occurring regulatory T cells expressing the IL-33 receptor, ST2 (161.1)
Authors: Mathews Lisa , Rosborough Brian , Thomson Angus , Turnquist Heth
Published: January 2023

Overview: Both IL-33 and its receptor, ST2, have been ascribed T helper type-2 (Th2) response promoting capacities. However, IL-33 increases splenic CD4+ forkhead box P3 (Foxp3)+ regulatory T cells (Treg) and post-transplant IL-33 monotherapy mediates a Treg-dependent prolongation of experimental cardiac allograft survival. As such, we addressed whether IL-33 directly targets Treg to support their expansion or suppressive function. Methods: The ability of recombinant IL-33 to facilitate anti-CD3/CD28-stimulated proliferation of BALB/c St2+/+ or St2-/- CD4+ CD25+ T cells was compared to IL-2. Following stimulation, Treg phenotype was defined by flow cytometry and their capacity to suppress naive T cell proliferation determined in bead-based suppression assays. Results: Following 3-4 days of culture, both cytokines promoted significant Treg proliferation vs. CD3/CD28 stimulation alone. However, IL-33 facilitated the expansion of a population ST2+ Foxp3+ cells that were absent from IL-2-treated cultures. Both IL-2 and IL-33-exposed Treg cultures exhibited suppressive activity. Conclusions: We identify the previously unreported capacity of IL-33 for direct expansion of Treg. Also, our data reveal that ST2, in addition to being expressed on Th2 cells, is found on an IL-33-expanded subset of suppressive Treg. These findings have significant implications for the therapeutic use of IL-33 to expand Treg, -cells critically important to tolerance and organ allograft survival.
PCNA: a novel cancer-associated ligand for the NK cell receptor NKp44 (162.10)
Authors: Brusilovsky Michael , Rosental Benyamin , Hadad Uzi , Campbell Kerry , Porgador Angel
Published: January 2023

Natural Cytotoxicity Receptors (NCRs: NKp30, NKp44, and NKp46) trigger NK cell-mediated lysis of tumors and virus-infected cells. Previous studies identified BAT3, B7-H6 and Vimentin as cellular ligands for NKp30 and NKp46, respectively. However, our knowledge of NCR-mediated recognition of cancer cells remains incomplete. NKp44 is expressed exclusively in primates and its expression is induced upon activation of NK cells. We have discovered that NKp44 directly recognizes Proliferating Cell Nuclear Antigen (PCNA), which is recruited within target cells to the NK immunological synapse. Over-expression of PCNA was found to inhibit NK cell cytotoxicity in an NKp44-dependent manner. Also, the cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) in NKp44 is crucial for PCNA-meditated NK cell inhibition. Since PCNA over-expression can promote cancer survival, our study reveals a novel immune evasion mechanism. Our results also further emphasize the role of nuclear proteins as ligands for Natural Cytotoxicity Receptors.
M2b monocytes predominantly appear in patients with advanced hepatocellular carcinoma (HCC patients) (162.29)
Authors: Asai Akira , Tsuchimoto Yusuke , Fukunishi Shinya , Tsuda Yasuhiro , Fukuda Akira , Kobayashi Makiko , Suzuki Fujio , Higuchi Kazuhide
Published: January 2023

In response to tumor environments, resident Mϕ differentiate into M1Mϕ or M2Mϕ. Recently, three different subsets of M2Mϕ (M2aMϕ, M2bMϕ and M2cMϕ) have been reported. Therefore, in this study, the properties and subsets of monocytes isolated from early and advanced HCC patients were characterized. CD14(+) monocytes were isolated from peripheral blood of HCC patients by Ficoll-Hypque sedimentation and magnetic beads. HCC patients, who were confirmed as carriers of hepatic cancer cells, were enrolled in this study. Monocytes (1 x 10(6) cells/ml) were cultured for 48 hrs and culture fluids harvested were assayed for cytokines by ELISA. Tumoricidal activity of monocytes against hepatic cancer cells was assessed using a standard chromium release assay. In the results, monocytes from advanced HCC patients were shown to be M2b monocytes, because they produced IL-10 (188 ± 21 pg/ml) and CCL1 (24 ± 0.6 ng/ml), and did not produce IL-12, CCL17 and CXCL13. Monocytes from early HCC patients were characterized as M1 monocytes, and healthy donor monocytes were determined as resident monocytes. Monocytes from early HCC patients killed syngeneic hepatic cancer cells (73 ± 7%), while monocytes from advanced HCC patients did not (10 ± 3%). These results indicate that advanced HCC patients are carriers of M2b monocytes and early HCC patients are M1 monocyte carriers. M2b monocytes detected in advanced HCC patients may play a role on the decreased host’s antitumor resistances.
Cancer immunoediting by the innate immune system in the absence of adaptive immunity (162.3)
Authors: Saddawi-Konefka Robert , O’Sullivan Timothy , Vermi William , Koebel Catherine , Arthur Cora , White J. Michael , Uppaluri Ravi , Andrews Daniel M. , Ngiow Shin , Teng Michele , Smyth Mark , Schreiber Robert , Bui Jack
Published: January 2023

Cancer immunoediting is the process whereby immune cells protect against cancer formation and sculpt the immunogenicity of developing tumors. The complete process of immunoediting requires both innate and adaptive immunity, but it remains unclear to what extent innate immunity alone is capable of immunoediting. To determine whether the innate immune system can edit tumor cells in the absence of adaptive immunity, we compared the incidence and immunogenicity of 3’methylcholanthrene-induced sarcomas in syngeneic wild-type, RAG2-/-, and RAG2-/-x γc-/- mice. We found that innate immune cells could indeed manifest cancer immunoediting activity in the absence of adaptive immunity. This activity required γc and IFNγ, which mediated the induction of M1 macrophages. M1 macrophages could be elicited by administration of CD40 agonists, thereby restoring editing activity in RAG2-/-x γc-/- mice. Our results suggest that in the absence of adaptive immunity, natural killer cell production of IFNγ induces M1 macrophages, which act as important effectors during cancer immunoediting.
Ongoing priming of naïve melanoma/melanocyte specific CD8 T cells occurs in hosts with vitiligo (P225) (162.6)
Authors: Byrne Katelyn , Steinberg Shannon , Zhang Peisheng , Turk Mary Jo
Published: January 2023

Vitiligo, the autoimmune destruction of melanocytes, correlates with improved survival in melanoma patients although it is often disregarded as a side effect of robust anti-tumor T cell responses. We have shown that antigen provided by vitiligo is required for long-lived CD8 T cell memory against melanoma-expressed self-antigens. However, the possibility that autoimmune melanocyte destruction itself may prime CD8 T cells able to cross-react against melanoma has not been investigated. Previously, we reported vitiligo development in 60% of mice depleted of regulatory CD4 T cells while bearing established B16 melanoma tumors. To probe the immunogenicity of vitiligo, we transferred naïve melanocyte-antigen specific pmel CD8 T cells in to actively depigmenting hosts. We find that CD8 T cell activation and proliferation occurs in an antigen-specific manner only in hosts with vitiligo. Furthermore, although the pmel cells do not produce IFN-γ, the cells produce Granzyme B and express LAMP-1 and P selectin ligand. Interestingly, although vitiligo-primed CD8 T cells appear strongly activated, the cells do not form robust memory populations in the presence of CD4 T regulatory cells. Ongoing studies aim to investigate the anti-melanoma capabilities of vitiligo-primed CD8 T cells. These studies will further enhance our understanding of the direct relationship between tumor immunity and autoimmunity, and the contributions of these cells in protecting against recurrent tumor growth.
Cholera toxin enhances IL17 production by CD4+ T cells via cyclic AMP signaling pathway (163.5)
Authors: Tsai Hsing-Chuan , Wu Reen
Published: January 2023

Cholera toxin (CT) is one of the most potent mucosal adjuvants. It has been reported to improve antigen presentation as well as suppress resting T cell activation. Recently, CT was also reported to indirectly increase the Th17 response through elevating IL-6 production from dendritic cells. This result suggests a vital role of CT in mucosal immunity. The goal of our study is to understand how cholera toxin modulates the response of CD4 T cells. We found that CT can directly enhance IL-17 production by CD4+ T cells but decrease IFN-γ, TNF-α, IL2, and IL-10 production. In addition, CD4 T cells were treated with different cAMP-elevating agents, including CT B subunit and PKA inhibitors. We found that the effect of cholera toxin on Th17 response goes through a PKA-independent but cAMP signaling dependent pathway. A more detailed study of cAMP signaling is required to fully understand how cAMP signaling controls this Th17 response. Furthermore, CT may also prevent Th17 cells from becoming Th17/Th1 cells which are more pathogenic in Th17-mediated diseases. Whether cholera toxin or cAMP signaling constrains Th17-mediated pathology will be examined.
Time-dependent IFN-γ and CXCL-10 production in tuberculosis patients after the establishment of clinical cure (164.5)
Authors: Silva Marcos , Massaro Júnior Vladimir , Machado Juliana , Castellano Lucio , Rodrigues Denise , Rodrigues Virmondes
Published: January 2023

Introduction: The immune response involved in the establishment of the clinical cure is not fully elucidated in human tuberculosis. The understanding of Th1 cytokines and chemokines role may drive the development of new therapeutic and diagnostic approaches. Objectives: The objective was to evaluate the IFN-γ and CXCL-10 production by BCG soluble antigen-stimulated PBMCs from patients with pulmonary tuberculosis. Methods: Blood samples were collected from 26 TB patients being 12 with active disease, 7 clinically cured patients with less than 12 months of treatment end and 7 with more than 12 months. PBMCs were cultured in RPMI medium alone or stimulated with 2ug/ml PHA or 10ug/ml BCG antigen. The IFN-γ and CXCL-10 production was determined in culture supernatants by ELISA. Results: The TB-treated patients showed significantly higher production of IFN-γ and CXCL-10 in all culture conditions than active disease patients. The augmented production of IFN-γ was mainly observed in clinically cured patients with more than 12 months of treatment. However, the CXCL-10 elevation was observed mainly on clinically cured patients with less than 12 months treatment. Conclusion: These results indicate that the potentiation of IFN-γ and CXCL-10 production occurs at different times after the establishment of clinical cure. This may suggest that the turn toward a Th1 immune response occur later, after clinical cure.
Failure of Rituximab in solid tumors is due to its inability to eliminate tumor evoked B regulatory cells (165.15)
Authors: Bodogai Monica , Lee Chang Catalina , Wejksza Katarzyna , Malchinkhuu Enkhzol , Chan Andrew , Biragyn Arya
Published: January 2023

We have previously shown that breast cancer generates regulatory B cells (tBregs) to convert Tregs and thereby to enable its metastasis. Thus, we decided to deplete tBregs using aCD20 antibody, a counterpart of the FDA approved strategy for the treatment of B cell malignancies (Rituximab). To our surprise, when tumor bearing mice were treated with αCD20 antibody, tumor progression was accelerated leading to increased lung metastasis. Thus, we hypothesized that tBregs may not be targeted by the antibody due to their low expression of CD20. Indeed, we found that B cell during their conversion to tBregs, downregulate their CD20 expression. Thus, tumor bearing mice treated with aCD20 antibody had enriched amounts of tBregs. We provide evidence that CD20 cannot be a target for the depletion of tBregs, explaining the failure of Rituximab to improve clinical outcomes in solid tumors. Instead, we developed an alternative strategy by in vivo targeting CpG through CXCR5 expressed by tBregs. We show that BLCArp-CpG inactivates tBregs, thereby reversing tumor metastasis. Taken together, suppressive B cells need to be controlled in solid tumors.
IL-2 withdrawal from human CD8+ T cell expansion in the presence of IL-7 enables retention of favorable functional and phenotypic characteristics for adoptive immunotherapy (165.2)
Authors: Ho Y. , Brooks Anna , Dunbar P.
Published: January 2023

Viral infection and reactivation of latent viruses are major causes of mortality and morbidity following haematopoietic stem cell transplantation. Adoptive immunotherapy with CD8+ T cells has proven efficacy in preventing or treating post-transplant viral infection, including therapy with resting T cells that undergo homeostatic expansion in vivo that follows transplantation. However T cells continuously expanded in vitro in IL-2 lose many characteristics associated with efficacy in vivo, and most IL-2-expanded cells die soon after infusion. Here we describe an alternative expansion protocol for human CD8+ T cells that only exposes the cells briefly to IL-2. Survival of the cells after IL-2 withdrawal relies on the continuous presence of IL-7, and the cells eventually come to rest in the cultures. In this system, timed exposure of naïve CD8+ T cells to IL-12 and IL-21 generates memory cells with competent effector functions upon rechallenge, specifically strong production of IFNγ, TNFα and IL-2. The memory cells generated also have acquired a favourable phenotype associated with early differentiation status by both cell surface markers (CCR7, CD62L, CD27 and CD28) and transcription factors (eg Tcf-1). Although our system has been piloted on the expansion of naïve CD8+ T cells, we propose that this alternative protocol could be used to generate resting antigen-specific memory cells with improved efficacy in preventing or treating viral disease in immunocompromised patients.
Induction of high levels of resistance to schistosome infection in buffalo vaccinated with a prime/boost regimen using a new delivery method. (166.14)
Authors: Li Changlin , Da'darah Akram , Li Yuesheng , Harn Donald
Published: January 2023

Schistosomiasis japonicum remains a serious zoonotoc disease of man and livestock, with water buffaloes accounting for about 75% of schistosome transmission to humans in China. A mathematical model of schistosome transmission suggests that a vaccine that induces 40% efficacy against infection, would significantly reduce transmission to humans. We tested the ability of SjC23, using four different vaccine delivery regimens, to induce the desired levels of efficacy in water buffalo. Comparing GLP-grade plasmid DNA Sj23-Hsp70 to our in-house produced DNA vaccine, we observed no differences in efficacy. Increasing the time between boosts from 1 to 3 months actually resulted in lower levels of efficacy, 37.2% and 31.5% compared with 41.2% and 45.8% adult worm burden and liver egg burdens respectively. Co-administration of an IL-12 plasmid DNA significantly enhanced SjC23 plasmid DNA vaccine efficacy. We then tested priming with SjC23 and SjCTPI plasmid DNAs, followed by a boost with recombinant SjC23 and rec SjCTPI, using our new vaccine delivery method. The prime/boost, new delivery method strategy yielded the highest levels of efficacy seen to date; 55% reduction in adult worms and 57% reduction in fecal egg hatching rates compared to levels in control vaccinated buffalo. Serological and immunological analyses are under way. We believe we can further enhance vaccine efficacy by priming with plasmid DNA vaccines delivered by electroporation.
Carbohydrate modification of novel polyanhydride nanoparticle adjuvants to promote pathogen-mimicking characteristics (166.5)
Authors: Phanse Yashdeep , Carrillo-Conde Brenda R. , Ramer-Tait Amanda , Roychoudhury Rajarshi , Pohl Nicola , Narasimhan Balaji , Wannemuehler Michael , Bellaire Bryan
Published: January 2023

An imminent need exists to design improved adjuvants and vaccines to control emerging and re-emerging diseases. An ideal vaccine will mimic the way in which a naturally occurring infection induces a robust immune response yet avoids the undesirable effects of disease. To accomplish this, new adjuvants must engage pattern recognition receptors on antigen presenting cells (APCs). In this work, we employ a novel strategy to enhance the adjuvanticity of polyanhydride nanoparticles (NP) composed of 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and 1,6-bis(p-carboxyphenoxy) hexane (CPH) by decorating their surface with the pathogen associated molecular pattern di-mannose (diM) to confer “pathogen-like” properties. Co-incubation of diM-NP with APCs elicited significant increases in surface expression of antigen presentation and co-stimulatory molecules and secretion of proinflammatory cytokines. An 800% increase in uptake of diM-NP was also observed. Furthermore, the mechanism of NP uptake was actin mediated and clathrin dependent, as is the case for many bacterial pathogens. Together, our data show that diM-NP possessed similar patterns of intracellular fate, persistence and APC activation as did live, pathogenic Y. pestis and E. coli. The innovative combination of polyanhydride NPs and di-mannose modification provides mechanistic insight into the properties required for next-generation adjuvants to safely mimic the immune stimulating activities of bacterial pathogens.
The role of mannose binding lectin (MBL) in co-infection of pandemic H1N1 and Streptococcus pneumoniae (168.14)
Authors: Ling Man To , Han Yan , Tu Wen Wei , Ho Pak Leung , Lau Yu Lung
Published: January 2023

Pandemic H1N1 (pdmH1N1) influenza A virus caused massive outbreak in 2009 and currently circulates in the community worldwide. Recent studies on this pandemic revealed that most of the deaths were resulted from secondary bacterial pneumonia caused by common bacteria found in upper respiratory tract, such as Streptococcus pneumoniae. Previous studies have shown that mannose binding lectin (MBL) exhibits anti-viral and anti-bacterial effects in vitro and in vivo. However, the immunomodulatory role of MBL during pdmH1N1/ S. pneumoniae co-infection has not been reported before. In this study, we aim to compare the severity of pdmH1N1/S. pneumoniae co-infection with pdmH1N1 infection alone, and to elucidate the functional implication of MBL during co-infection. Our in vitro preliminary data showed that recombinant MBL could successfully bind to immobilized pdmH1N1 in a dose-dependent manner. However, recombinant MBL was found to be incapable of binding immobilized S. pneumoniae. WT and MBL KO mice were infected with a sublethal dose of pdmH1N1 and S. pneumoniae. Co-infection resulted in a more severe disease phenotype and mortality in mice. However, no significant difference in weight loss and survival rate was observed between WT mice and MBL KO mice. Taken together, these data suggest that MBL may play a more vital role in modulating the first pdmH1N1 infection before subsequent bacterial infection and may attenuate the severity of the co-infection.
The molecular mechanism of dengue virus-induced IL-10 production in monocytes (168.15)
Authors: Lin Chiou-Feng , Chuang Yi-Jui , Lin Yee-Shin , Wan Shu-Wen , Chen Chia-Ling
Published: January 2023

Dengue virus (DV)-caused anti-inflammatory IL-10 production is much higher in patients with severe dengue hemorrhagic fever and dengue shock syndrome than those with mild dengue fever; however, the regulation of IL-10 is still unclear. Here we showed that DV infection significantly increased IL-10 production and then caused suppressor of cytokine signaling 3 expression in human monocytic THP-1 cells. In DV-infected cells, activation of CREB determined IL-10 expression largely through cAMP-dependent protein kinase A (PKA)- and PI3K-regulated manners. Furthermore, sequential activation of cAMP and PKA facilitated PI3K/PKB signaling. Notably, DV infection caused glycogen synthase kinase (GSK)-3β inactivation in a PKA/PI3K-regulated manner and inhibiting GSK-3β considerably increased DV-induced IL-10 production following CREB activation. However, blocking the well-known DV receptors, including CD209 (DC-SIGN), CD29 (integrin β1), and CD61 (integrin β3), did not inhibit DV-induced IL-10. These results show a potent molecular basis for DV infection-induced IL-10 production in monocytes. The involvement of receptors related to signaling axis of cAMP, PKA, PI3K, PKB, GSK-3β, and CREB needs further investigation.
Mast cell-dependent inflammatory response during influenza A virus infection (168.31)
Authors: Graham Amy , Zickovich Julianne , Hilmer Kimberly , Obar Joshua
Published: January 2023

The lung is protected from pathogens by alveolar epithelial cells, tissue resident alveolar macrophages, dendritic cells, and mast cells. The role of the mast cell has been under explored during severe pulmonary viral infections. In mice that lack functional mast cells, pathological immune responses are altered depending on the influenza strain. We found that both A/WSN/33 (WSN) and A/PR/8/34 (PR8) influenza viruses generate significant immunopathology in C57BL/6 mice, but only WSN induced pathology was mast cell dependent. To assess the differences in mast cell dependent inflammation response due to influenza strain, we examined the ability of each strain to activate in vitro-derived bone marrow cultured mast cells (BMCMC). We found that WSN, but not PR8, influenza virus activated BMCMC to produce inflammatory cytokines/chemokines, anti-viral chemokines, and degranulate. Importantly, a recombinant PR8 virus that expresses the WSN hemagglutinin (HA) glycoprotein instead of its own could induce cytokine production in BMCMC, demonstrating an important role for the HA glycoprotein for mast cell activation. Mast cell infection and viral RNA synthesis and export were necessary for mast cell activation and occurred in a MAVS-dependent mechanism. Thus, we have identified a unique inflammatory cascade which could be targeted to limit morbidity following infection with influenza virus.
Elevated levels of circulating galectin-9, a novel immune-modulator, in acute dengue virus Infection reflects disease severity (170.18)
Authors: Chagan-Yasutan Haorile , Ndhlovu Lishomwa C. , Lacuesta Talitha Lea , Leano Prisca Susan A. , Telan Elizabeth Freda O. , Niki Toshiro , Hirashima Mitsuomi , Dimaano Efren M. , Hattori Toshio
Published: January 2023

Following a dramatic rise in incidence in recent decades, Dengue virus infection (DENV) is now a significant burden of disease in the tropics and major international public health concern. Currently no treatment or effective vaccine exists. The β-Galactoside-binding lectin, Galectin-9 (Gal-9), functions as pro-apoptotic agent and inducer of pro-inflammatory cytokines from T cells. Here, we sought to evaluate the role of Gal-9 in the pathogenesis of acute DENV in patients presenting with self-limiting dengue fever (DF) and more serious life-threatening dengue hemorrhagic fever (DHF). Blood samples were obtained from 65 patients (mean age: 23±4yrs) hospitalized with DENV from the San Lazaro Hospital, Manila, Philippines in 2010. Plasma Gal-9 was measured by an ELISA method at the critical phase (day4-5) and recovery phase (day7-8) of infection. Our results show that during the critical phase, Gal-9 levels were markedly elevated in DF (1758±1047pg/ml) and DHF(2730±1848pg/ml) compared to uninfected (363±280pg/ml) (p<0.05) controls. These levels of Gal-9 were the highest ever observed in humans. During the recovery phase, Gal-9 levels significantly declined (1170pg/ml and 1140pg/ml) (p<0.05) from peak levels in DF and DHF, respectively, but not to levels observed in uninfected donors. The above findings suggest that Gal-9 levels tracks disease severity during acute DEN, potentially mediate immuno-pathology associated with DHF, and may serve as an indicator of recovery from DENV.
Crucial role for IPS-1 signaling in mediating anti-viral responses and clearance of Respiratory Syncytial Virus (170.4)
Authors: Demoor Tine , Petersen Bryan , Morris Susan , Berlin Aaron , Schaller Matthew , Lukacs Nicholas
Published: January 2023

Upon replication of Respiratory Syncytial Virus (RSV), double-stranded viral RNA can be detected in the endosomes via Toll-like receptor 3 (TLR3) signaling or by the cytoplasmic helicases retinoic acid inducible gene I (RIG-I) and melanoma differentiation-associated gene (Mda5). The latter interact downstream with IFNβ promoter stimulator (IPS-1). We previously demonstrated that TLR3 deficiency worsens RSV-induced lung pathology, yet leaves viral clearance intact. The present study examined the importance of IPS-1 signaling for anti-viral responses in primary cultures of alveolar epithelial cells (AECs), in dendritic cells (DCs), and macrophages (mΦ), as well as in vivo using IPS-1 knockout (KO) mice. In AECs, RSV induced IFNβ, IL6, CXCL1, CCL2 and CCL5 in an IPS-1 dependent manner. In DCs, the expression of IFNβ and CCL2 also required IPS-1. Conversely, RSV-induced cytokine and chemokine expression was enhanced in IPS-1 KO mΦ, while T-cells of infected IPS-1 KO mice showed a predominant Th1 profile with increased IFNγ. Plaque assays revealed higher RSV titers in IPS-1 deficient mice, accompanied by more severe granulocytic infiltration. Our data suggest a significant contribution of the IPS-1 pathway to the RSV-induced type I interferon response and inflammatory cytokine/chemokine production. Future experiments, using bone marrow chimeras, will help to elucidate the role of IPS-1 in both the structural and hematopoietic mediated response to viral infection.