bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytoskeletal stiffness controls the threshold of T cell activation (IRM10P.747)
Authors: Butte Manish , Bruce Marc , Thauland Timothy
Published: January 2023

T cells require a dynamic cytoskeleton to effectively scan antigen-presenting cells and become fully activated. To study the function of cytoskeletal changes as they occur in real time during T cell activation, we modified an atomic force microscope to deliver antigen via the cantilever, simultaneously ligating T-cell receptors (TCRs) and measuring changes in cytoskeletal mechanics. We show that antigenically naïve T cells are mechanically stiff and their cytoskeletal structures undergo rapid relaxation at the site of TCR ligation. In contrast, antigen-experienced memory and effector T cells are already soft prior to TCR triggering. We found that cytoskeletal relaxation is due to depolymerization of actin, is proportional to antigen dose, and requires proximal TCR signaling pathways but not ICAM-1:LFA-1 interaction. We identified pathways controlling the stiffness of naïve T cells, and blocking these pathways resulted in naïve T cells become soft even prior to TCR triggering. Moreover, these soft yet antigen-inexperienced T cells showed a reduced threshold for allo- and antigen-specific activation compared to controls. Our results highlight the functional importance of cytoskeletal stuctures in tuning T cell responses to spatially localized signals from APCs.
Down-modulation of tumor suppressor p53 by T cell receptor signaling is critical for antigen-specific T cell responses (IRM10P.737)
Authors: Watanabe Masashi , Moon Kyung duk , Vacchio Melanie , Hathcock Karen , Hodes Richard
Published: January 2023

We report here that antigen-specific proliferative responses of naïve and memory CD4 T cells require the down-modulation of tumor suppressor p53. In the absence of TCR signal, IL-2 induces a sustained increase in p53 protein, which prevents proliferative responses despite strong signaling through the IL-2 receptor. In contrast, TCR signaling results in early termination of p53 protein expression by decreasing p53 mRNA as well as by strong transcriptional induction of the p53-regulating protein Mdm2. Down-modulation of p53 in response to antigen stimulation is in fact critical for antigen-specific T cell proliferation; and preventing p53 degradation by inhibiting Mdm2 results in sustained p53 protein levels and prevents antigen-specific T cell proliferation. These studies elucidate a critical role of p53 as a negative regulator of T cell proliferation. It is the termination of p53 elevation by TCR signaling that allows proliferative responses to occur, enforcing antigen specificity.
Phosphatidylcholine as a metabolic cue for determining B cell fate and function (LYM6P.774)
Authors: Barrington Robert , Solodushko Viktoriya , Aragon Ileana , Brewer Joseph
Published: January 2023

In activated B cells, increased production of phosphatidylcholine (PtdCho), the most abundant cellular phospholipid, is handled primarily by the CDP-choline pathway. Previous studies revealed that CD19Cre-mediated deletion of CTP:phosphocholine cytidylyltransferase α (CCTα), the rate-limiting enzyme in the CDP-choline pathway, led to augmented IgM secretion and reduced IgG production, consistent with defective germinal center reactions. To more specifically assess the influence of PtdCho on B cell fate during germinal center and long-term humoral responses, we examined immune responses in Cγ1Cre CCTαflox mice. Serum IgG1 was undetectable in naïve Cγ1Cre CCTαflox mice, while levels of IgM and other IgG subclasses were similar in naïve Cγ1Cre CCTαflox and control mice. Following immunization with T cell-dependent antigen NP-KLH, control mice predictively generated high titer IgG anti-NP. In contrast, IgG anti-NP titers were markedly reduced in immunized Cγ1Cre CCTαflox mice. Interestingly, though antigen-specific IgM responses were comparable between Cγ1Cre CCTαflox and control mice, the IgM anti-NP response in Cγ1Cre CCTαflox mice failed to increase in affinity. Lastly, the frequency of antigen-experienced NP-specific B cells and antibody-secreting cells (ASC) were both reduced in Cγ1Cre CCTαflox mice compared with controls. These data indicate that PtdCho plays a pivotal role in the generation and persistence of both germinal center B cells and ASC.
First mitotic division programs naïve B cell for successive cell divisions (LYM6P.778)
Authors: Singh Amit , Zhang Yongqing , Wood William , Becker Kevin , Sen Ranjan
Published: January 2023

Transient activation via the B cell receptor (BCR) primes naïve B cells to respond more efficiently to CD40 signals, resulting in increased cell division over a 48hr time course. At this time point approximately 40% of primed B cells had undergone at least one division, as compared to 15% cells activated by anti-CD40 antibody alone. Cells that had divided once continued through several additional division cycles in the absence of exogenous stimuli, whereas cells that had not divided after the first 48hr required renewed anti-Ig signals to induce proliferation. These observations demonstrate that successful negotiation of the first mitosis make mature B cells permissive for signal-independent proliferation. Characteristics of once divided cells that give them this unique property will be presented and the implications for physiological responses of B cells will be discussed.
Dynamics of B cell subsets within the CNS during viral encephalomyelitis (LYM6P.779)
Authors: Disano Krista , Phares Timothy , Bergmann Cornelia
Published: January 2023

Central nervous system inflammation induced by viral infection and autoimmune disease results in the accumulation of plasmablasts. During neurotropic coronavirus infection, CD138+CD19+ plasmablasts are essential in preventing viral reemergence after initial T cell mediated control of infectious virus. This study characterizes the temporal dynamics and precursor relationships between B cell subsets within the infected central nervous system. Flow cytometry revealed that CD138- B cells recruited early displayed a naïve or early activated IgD+IgM+ phenotype. This population progressively declined coincident with an increase of IgD-IgM+ and isotype switched memory B cells and plasmablasts, suggesting early B cells differentiate locally or are replaced by newly recruited, more differentiated B cells. Ki-67 expression by CD138- B cells revealed proliferation in IgD+IgM+ B cells was minimal, while proliferation was substantially elevated in IgDloIgM+ and IgD-IgM+ cells, consistent with recruitment of peripherally proliferating B cells rather than local differentiation of naïve B cells. Nevertheless, a transient capacity of IgM+ and IgD+ B cells to enter the parenchyma early in infection suggests they may have an antibody independent role during central nervous system inflammation.
Long-term bone marrow IgM-producing plasmablasts are derived during acute infection and are maintained by active proliferation (LYM6P.767)
Authors: Papillion Amber , Winslow Gary
Published: January 2023

We have previously characterized a population of IgM-secreting B cells in the bone marrow (BM) of mice infected with the intracellular bacterium Ehrlichia muris (J. Immunol. 186:1011). The BM cells are responsible for IgM production, which is sufficient to maintain long-term immunity to fatal infection. In the present study, we have investigated the ontogeny and maintenance of the B cell population. We utilized AID-Cre-ERT2 X Rosa26loxP-EYFP mice (Nat. Immunol. 10:1292) to irreversibly mark activation-induced cytidine deaminase (AID)-expressing B cells. Following tamoxifen administration on day 10 post-infection, the time of peak spleen CD11c-positive plasmablast response, we detected EYFP+ CD138+ IgM+ BM cells for at least as long as 100 days post-infection; the EYFP+ cells were maintained at frequencies as high as 25% of the BM IgM+ B cells. These data indicated that the BM B cells were likely derived from T cell-independent spleen plasmablasts, and constituted a long-lived population. BrdU incorporation studies also demonstrated that the majority of the IgM+ cells were actively proliferating, indicating that B cells were plasmablasts. Finally, antibody-mediated blocking of CD40:CD40L signaling during acute infection did not impact the generation of the BM plasmablasts, indicating that the plasmablasts were likely generated in the absence of T cell help. These findings underscore the importance of BM IgM plasmablasts in long-term humoral immunity and host defense.
The macrophage-derived proteins murine RELMα and human resistin regulate host immunity to helminth infection (MPF3P.801)
Authors: Nair Meera
Published: January 2023

Th2 cells are essential for protective immunity to helminths but also contribute to chronic inflammation. Here we identify the REsistin-Like Molecules RELMα and Resistin as critical mediators of this balance between helminth Th2-mediated immunity and inflammation in both murine infection and human studies. Following infection with Nippostrongylus brasiliensis, we observed significant macrophage expression of RELMα in the lungs. When RELMα-/- mice were infected with N. brasiliensis, there were increased Th2-type responses, accelerated worm expulsion and exacerbated lung inflammation compared to wild-type mice, implicating a role for RELMα in dampening Th2 immune responses. Selective depletion of macrophages in RELMα-/- mice ameliorated N. brasiliensis-induced chronic lung inflammation, suggesting that RELMα modulates inflammation through effects on macrophages. In new translational studies, we investigated the function of human Resistin (hResistin) which shares sequence homology with RELMα. Employing transgenic mice that express hResistin, we observed increased macrophage expression of hResistin in N. brasiliensis-infected lungs. Moreover, hResistin inhibited Th2 immune responses and immunity to N. brasiliensis. Finally, we observed a strong correlation between parasite number and serum Resistin levels in a population of helminth-infected patients (n=46). Taken together, these studies identify a new RELM-mediated regulatory pathway in helminth immunity and inflammation.
Effect of CCL1 antisense oligodeoxynucleotides (ODN) on MRSA infection in subacutely burned mice (SB-mice) (MPF3P.810)
Authors: Kobayashi Makiko , Ito Ichiaki , Nishiguchi Tomoki , Herndon David , Suzuki Fujio
Published: January 2023

Staphylococcus aureus is frequently isolated from subacutely burned patients, and these infections easily develop into severe sepsis. M2bMφ have been characterized as the responsible cells on the increased susceptibility of SB-mice to staphylococcal infections, and SB-mice are carriers of M2bMφ. In this study, we tried to protect SB-mice (mice 10 to 30 days after burn injury) from a lethal dose infection of MRSA using CCL1 antisense ODN (an inhibitor of M2bMφ). Mice, 14 days after burn injury (20% total body surface area 3rd degree burns), were infected with 2 x 106 CFU/mouse of MRSA. Then, these mice were treated s.c. with 10 μg/mouse of CCL1 antisense ODN twice a day for 3 days (treated mice). As control mice, SB-mice were treated with scrambled ODN. The protective effect of the ODN against MRSA infection was evaluated by a reduction in the mortality rates of treated mice as compared with that of control mice. Also, bacterial growth in various organs of treated mice was compared with those of controls. In the results, all control mice died within 7 days of MRSA infection, while all treated mice were still alive after 7 days with the same infection. Bacteria grew in the liver, spleen and kidneys of control mice. However, the pathogen did not grow significantly in these organs of treated mice. These results indicate that sepsis caused by a lethal dose of MRSA infection in SB-mice is controlled by treatment with CCL1 antisense ODN.
Early in vivo infection dynamics in non-human primates infected with Mycobacterium tuberculosis (MPF3P.813)
Authors: Cadena Anthony , Martin Constance , Fortune Sarah , Flynn JoAnne
Published: January 2023

Tuberculosis (TB) continues to pose a significant health risk to morbidity and mortality worldwide. The vast majority of individuals infected with M. tuberculosis, the causative agent of TB, contain the infection as clinically asymptomatic latent TB. A small minority of people do not and either present with primary active disease or reactivate over their lifetime. By examining early events in infection at the local lesion level, we aim to uncover some of the basis for the observed spectrum of TB disease and host outcome. Using genetically barcoded M. tuberculosis, serial PET/CT lesion imaging, and tools to analyze each granuloma at the microbial and immunological level, we are tracking local lesion development and dissemination at various stages of infection in cynomolgus macaques. Background studies have revealed that granulomas are dynamic entities that largely arise from single bacteria. We hypothesize that early interactions between M. tuberculosis and host immune components contribute to lesion heterogeneity and that these initial events influence infection outcome. We performed a small reinfection experiment with M. tuberculosis barcoded strains. Our data suggests that primary infection limits secondary infection lesion development and dissemination. Further analysis of this study is ongoing. Probing the mechanisms that underlie the human TB spectrum will reveal crucial stages of infection that can be exploited for novel therapeutic design.
The TLR2 signaling pathway is activated by sporozoites and suppresses intrahepatic parasite development (MPF3P.815)
Authors: Xu Wenyue , Zheng Hong , Tan Zhangping , Zhou TaoLi
Published: January 2023

TLRs (Toll-like receptors) play an important role in the initiation of innate immune responses against invading microorganisms. Although several TLRs have been reported to be involved in the innate immune response against the blood-stage of malaria parasites, the role of TLRs in the development of the pre-erythrocytic stage is still largely unknown. Here, we find that sporozoite lysate can significantly activate TLR2. Further studies show that sporozoite lysate can be recognized by either TLR2 homodimers or TLR2/1 and TLR2/6 heterodimers and induce macrophages to release proinflammatory cytokines in a TLR2-dependent manner. Interestingly, the TLR2 signaling pathway can significantly suppress the development of the pre-erythrocytic stage, and the administration of TLR2 agonists greatly reduced the liver parasite load of mice challenged with sporozoites. Additionally, the observed higher level of parasite burden in TLR2-/- mice is found to be closely associated with a reduction in proinflammatory cytokines in the liver.Therefore, we provide the first evidence that sporozoites can activate the TLR2 signaling pathway, which in turn significantly inhibits the intrahepatic parasites. This may not only help us to understand the mechanism of the innate immune response against pre-erythrocytic stage of malaria parasite but may also provide us with novel clues to design preventive anti-malaria therapies.
The protective role of macrophages in Francisella tularensis infection (MPF3P.819)
Authors: Steiner Donald , Furuya Yoichi , Metzger Dennis
Published: January 2023

Francisella tularensis causes lethal pneumonia following infection of the lungs. F. tularensis is considered to be an intracellular pathogen that targets macrophages for replication. We therefore hypothesized that depletion of alveolar macrophages would impede bacterial replication and improve host survival. However, it was found that macrophages are essential for survival of F. tularensis infection and depletion of these cells reduced survival rates following infection. Mice with a macrophage-specific defect in interferon gamma (IFN-γ) signaling (MIIG mice) were also highly sensitive to infection, exhibiting reduced survival rates, reduced mean time to death, and increased bacterial burdens in lungs, liver, and spleen relative to wild-type mice. We have previously found that treatment with exogenous interleukin 12 (IL-12) protects against F. tularensis infection; this protection was lost in MIIG mice. MIIG mice also exhibited reduced neutrophil recruitment to the lungs following infection. Systemic neutrophil depletion was found to render mice highly sensitive to pneumonic F. tularensis infection. Furthermore, IL-12-mediated protection required NADPH activity. These results indicate that lung macrophages serve a critical protective role in pneumonic F. tularensis infection. Macrophages require IFN-γ signaling to mediate protection, which ultimately results in recruitment of neutrophils to further aid in survival from infection.
Mycobacterium tuberculosis early secreted antigenic target of 6 kDa (ESAT-6) induces IL-6 production by macrophages via STAT3 (MPF3P.820)
Authors: Jung Bock-Gie , Wang Xisheng , Yi Na , Barnes Peter , Samten Buka
Published: January 2023

Early secreted antigenic target of 6 kDa (ESAT-6) of Mycobacterium tuberculosis (Mtb) is a major virulence factor. Mtb-induced proinflammatory cytokine IL-6 inhibits macrophage responses to IFN-γ and contributes to the survival of Mtb. To explore the role of ESAT-6 in Mtb infection, we studied if ESAT-6 induces IL-6 production by macrophages. Incubation of mouse bone marrow-derived monocytes (BMDM) with ESAT-6 induced IL-6 production in dose and time dependent manner. However, culture filtrate protein of 10 kDa (CFP10) and antigen 85A (Ag85A) of Mtb did not induce IL-6 production, suggesting a specific effect of ESAT-6 on IL-6 production. Likewise, ESAT-6 but not CFP10 and Ag85A induced IL-6 secretion by human peripheral blood derived monocytes. Polymyxin B inhibited LPS but not ESAT-6 induced IL-6 secretion by macrophages refuting the possible role of LPS contamination in ESAT-6. ESAT-6 but not Pam3CSK4, a TLR2 agonist, stimulated IL-6 production by TLR2 knockout BMDM, indicating that this effect of ESAT-6 is TLR2 independent. To explore the signaling pathway of this, we examined the role of STAT3 since ESAT-6 induced activation of STAT3 in BMDM. Preincubation of cells with Stattic and S31-201, STAT3 inhibitors, but not rapamycin and DMSO (vehicle) inhibited ESAT-6 induced IL-6 expression by BMDM both at mRNA and protein level in a dose dependent manner without affecting cell viability. Thus, our results suggest that ESAT-6 induces IL-6 production in macrophages via STAT3.
Characterization of signaling pathways activated by helminth antigens in macrophages (MPF3P.808)
Authors: Valderrama-Carvajal Hector Francisco , Martínez-Saucedo Diana , Romero-Diaz Monica , Ruiz-Azuara Lena , Padilla-Viveros America , Padierna-Mota Laura , Canale-Zambrano Juan Carlos , Terrazas Luis I.
Published: January 2023

Helminths parasites modulate or escape their host’s immune response by different molecular and cellular strategies. One of these is through the interaction of different molecules from their surface with cellular membrane elements of immune cells, modulating signaling pathways. These parasites trigger, control and modulate the early response of macrophages and dendritic cells (DC). The aim of this study is to elucidate the macrophage signaling pathways that are activated in response to a Helminth Parasitic Antigen Fractions (HPAFs) in vitro. Our data show that HPAFs have a biological effect on macrophages. WB analysis using specific anti-phospho-antibodies, showed that the HPAFs can signalize in a time dependent manner. In addition, immunofluorescence experiments showed that HPAFs promote a significant change in macrophages morphology. Different reports indicate that the chemoattraction and change of morphology of the macrophages is an essential step in the host response to different infections. We evaluated the effect of HPAFs on the transcriptional regulation of downstream target genes. Our PCR studies showed that HPAFs-stimulated macrophages could regulate downstream target genes such as TLR-4, Arg-1, Ym-1 and iNOS, showing that HPAFs regulate important signaling pathways in macrophages. Our results suggest that HPAFs may contain molecules that could interact with membrane molecules, activating signaling pathways that are involved in the regulation of the immune response.
A Salmonella-encoded microRNA-like RNA facilitates bacterial invasion and intracellular replication via suppressing host cell inducible nitric oxide synthase (MPF3P.809)
Authors: Gu Hongwei , Zhang Tianfu , Li Donghai , Liu Fenyong , Zhang Chen-Yu , Zen Ke
Published: January 2023

Salmonella has developed a sophisticated machinery to promote virulence and survival. Here we show that Salmonella can hijack mammalian cell non-classical miRNA processing machinery to further process their non-coding RNAs into miRNA-like RNAs that modulate host cell function. Using Solexa deep sequencing, we detected a panel of 19-24nt Salmonella-derived ncRNA fragments in human colonic epithelial HT-29 cells following Salmonella infection. The fragment with the highest copy number, Sal-1, was validated by qRT-PCR and northern blot analysis. With seven copies in the S. enteritidis genome, Sal-1 has a stem structure-containing precursor with a free energy of ΔG= -26.1 kcal/mol. The processing of the Sal-1 “primary” /“precursor” to the mature Sal-1 in Salmonella-infected cells is Dicer-independent but Argonaute 2-dependent. Functionally, Sal-1 targets the expression of epithelial cell inducible nitric oxide synthase (iNOS) and thus reduces the NO production and bacterial resistance of host cells. Expression or depletion of Sal-1 in mouse colonic epithelium significantly enhanced or reduced Salmonella infection in mice, respectively. Finally, the enhancement of Salmonella infection in mice by Sal-1 was completely abolished by expressing a mutant iNOS isoform that had normal NO generation but could not be targeted by Sal-1. Our results show for the first time that Salmonella can produce functional miRNA-like RNAs, which may represent a novel class of virulence factors.
Altered inactivation of commensal LPS due to acyloxyacyl hydrolase deficiency in colonic dendritic cells impairs mucosal Th17 immunity (MUC4P.825)
Authors: Janelsins Brian , Datta Sandip
Published: January 2023

Interleukin 17 (IL-17)-secreting CD4+ helper T (Th17) cells are essential for host defense at mucosal surfaces, and dysregulation can result in autoimmunity. Exposure to microbial products such as lipopolysaccharide (LPS) can affect the ability of dendritic cells (DC) to polarize Th17 cells. Acyloxyacyl hydrolase (AOAH) is a mammalian enzyme expressed by antigen (Ag)-presenting cells that inactivates LPS in host tissues. We hypothesized that inactivation of intestinal-derived LPS by AOAH influences the ability of DC to prime Th17 cells. We found that LPS-containing microbiota augmented the polarization of Ag-specific Th17 cells, and identified CD103+CD11b+ colonic DC with the unique capacity to express AOAH and bias Th17 cells. Compared to wild-type, these Aoah-/- colonic DC produced less IL-6, which resulted in diminished Ag-specific Th17 polarization and increased regulatory T cell induction in vitro. Oral administration of LPS led to reduced IL-6 production from CD103+CD11b+ colonic DC in Aoah-/- mice in comparison to Aoah+/+ mice, resulting in an abrogated Ag-specific Th17 response in the gut after mucosal immunization that could be rescued by systemic delivery of recombinant IL-6. These data identify the ability of AOAH to modulate microbiota signals that drive mucosal Th17 polarization, and suggest that host pathways to handle microbiota-derived products may be targeted to modulate Th17 responses in the context of inflammatory disorders or infection at mucosal surfaces.
Neutrophils negatively regulate induction of mucosal IgA antibody responses to sublingual immunization (MUC4P.835)
Authors: Jee Junbae , Kim Eunsoo , Bonnegarde-Bernard Astrid , Iwakura Yoichiro , Cormet-Boyaka Estelle , Boyaka Prosper
Published: January 2023

Secretory IgA antibody plays an important role in the protection of mucosal surfaces against microbial pathogens. Induction of the IgA antibody has been known to be largely dependent upon the cytokine milieu. However, the importance of innate immune cell subsets for IgA responses has not been fully appreciated yet. We investigated the role of myeloid cell-subsets on IgA responses to sublingual immunization. In contrast with cholera toxin as an adjuvant which promotes IgA responses, sublingual application of the Bacillus anthracis edema toxin (EdTx) to C57BL/6 mice transiently increased the frequencies of neutrophils in sublingual tissue and cervical lymph nodes (CLNs), and promoted antigen-specific serum IgG, but not serum or mucosal IgA responses. However, EdTx as an adjuvant failed to increase frequencies of neutrophils in sublingual tissue and CLNs of mice lacking IKKβ in myeloid cells (IKKβΔMye), and these mice developed antigen-specific mucosal IgA responses. Interestingly, depletion of neutrophils in C57BL/6 mice promoted antigen-specific fecal and serum IgA responses, similar to those of IKKβΔMye mice, and this effect was associated with increased frequencies of surface IgA expressing plasma cells, α4β7+CCR9+ B cells in lamina propria and number of Peyer’s patch. Our results show that the presence of neutrophils at mucosal inductive sites negatively influences induction of IgA responses and underlying mechanisms are under investigations.
Commensal microbiota and food antigens are important factors in the regulation of IgE production in mice (MUC4P.848)
Authors: Hong Sung-Wook , Kim Kwang-Soon , Surh Charles
Published: January 2023

IgE is one of key modulators in the pathogenesis of allergy by inducing FcR-mediated activation of mast cells and basophils. Production of IgE is regulated by commensal microbiota, as it is normally undetectable in conventional mice, but highly elevated in germ-free mice shortly after weaning into solid food. The mechanism behind IgE production in germ-free mice is poorly understood, but could be a consequence of dysregulated immune responses to Ags in the food. To address this idea, germ-free mouse pups were weaned into an antigen-free elemental diet comprised of essential amino acids, vitamins and minerals. Strikingly, neither these mice nor their offsprings, designated as antigen-free mice, possessed detectable levels of serum IgE. Moreover, feeding adult germ-free mice with antigen-free diet reduced the serum IgE level, indicating continued presence of food is required to sustain high amount of IgE production. As expected, antigen-free mice weaned into normal solid food diet produced high levels of IgE. To determine the age-dependency of commensal microbiota in IgE production, germ-free mice were conventionalized at various ages, starting from 4 wks. Colonization of commensal microbiota at 4 wks of age prevented high levels of serum IgE, but not for older mice. These results suggest that food Ags by default induce a strong IgE response that is suppressed by the commensal microbiota.
Metabolites from commensal microbes are closely associated with maintaining mucosal homeostasis through modulation of innate lymphoid cells in Peyer’s patch (MUC4P.849)
Authors: Jang Yong-Suk , Kim Sae-Hae , Lee Ha-Yan
Published: January 2023

It was characterized that the innate lymphoid cells (ILCs) are a group of immune cells closely associated with immune functions such as lymphoid organogenesis, tissue remodeling, antimicrobial immunity, and inflammation through cytokine-mediated cell regulation. ILC subsets can be categorized into three groups based on cytokine-secreting patterns. Group 1 ILCs secrete IFN-γ. Group 2 produces type 2 cytokines dependent on GATA-3. Group 3 represents all RORγt+ ILC subtypes secreting IL-17 and/or IL-22. In mucosal immunity, RORγt+ ILCs play an essential role in early protection against enteric pathogen infections, although its regulation by mucosal environment is unknown. In this study, we suggest that butyrate, one of commensal microbe-derived metabolites, as a modulator of PP ILCs, because it is known that butyrate treatment closely associated with the protection of bacterial infection or DSS-induced colitis. In order to understand the mechanism underlying the proposed role of butyrate, we monitored the expression pattern of each transcription factors and cytokines in PP ILCs after oral administration of sodium butyrate in SPF and antibiotics-treated mice. To understand the regulating activity of PP ILCs induced by butyrate in mucosal immunity, we try to understand the regulation of DSS colitis in the same mice. Collectively, this study suggests that regulation of ILCs by butyrate in gastrointestinal tract was closely associated with maintaining mucosal homeostasis.
Microbial driven TLR5-dependent signaling modulates distal malignant progression through tumor-promoting inflammation (MUC4P.828)
Authors: Rutkowski Melanie , Stephen Tom , Allegrezza Michael , Svoronos Nikolaos , Perales-Puchalt Alfredo , Tesone Amelia , Nguyen Jenny , Tchou Julia , Conejo-Garcia Jose
Published: January 2023

The dominant R392X TLR5 polymorphism abrogates flagellin responses in >7% of humans. We report that TLR5 recognition of commensal bacteria at intestinal surfaces influences malignant progression at distal (extra-mucosal) locations through the initiation of tumor-promoting inflammation. In TLR5-responsive mice, IL-6 is up-regulated through an autocrine loop in IL-6 reactive tumor cells, resulting in increased mobilization of MDSC and augmented tumor growth, compared to TLR5-deficient controls. In contrast, TLR5-deficient mice systemically up-regulate IL-17, which augments tumor growth only in the presence of IL-6 unresponsive tumors. Notably, differences in tumor growth are completely abrogated following depletion of commensal bacteria. Most importantly, similar differences in inflammation and malignant progression are recapitulated in TLR5-responsive/unresponsive patients with extra-mucosal tumors. Our results demonstrate that TLR5 signaling at mucosal surfaces, which is dependent upon commensal microbiota, significantly influences tumor progression at extra-mucosal locations through systemic tumor-promoting inflammation. These results have implications for the large proportion of individuals that harbor the common genetic polymorphism in TLR5 and demonstrate that the systemic response to tumors outside of the intestines is in part mediated through TLR5-signaling.
Resident memory γδ T cells orchestrate response to secondary oral Lm infection (MUC4P.829)
Authors: Romagnoli Pablo , Sheridan Brian , Pham Quynh-Mai , Khanna Kamal , Lefrançois Leo
Published: January 2023

Intestinal mucosa shapes the immune system by interacting with commensal bacteria while preventing the invasion of bacterial pathogens. In a physiological model of oral Listeria monocytogenes (Lm) infection, we have previously reported the generation of Lm-elicited protective memory Vγ4 Vδ1 T cells that reside in lamina propria and mesenteric lymph nodes (mln). Here we tested the hypothesis that Lm-elicited γδ T cells drive the accelerated secondary immune response to oral Lm infection in the mln. We show that memory γδ T cells secreted IL-17A and IFNγ in vivo and formed clusters with neutrophils that surrounded Lm aggregates in the mln 1 day after secondary oral Lm infection (d.p.r.). Neutralizing IL-17A disrupted cluster formation and neutrophil recruitment leading to delayed bacterial clearance. Cluster formation was also abrogated by forced gamma delta TCR downregulation. Together, these observations demonstrate that IL-17A, secreted by Lm-elicited memory γδ T cells, was essential for both recruitment of neutrophils to the mln, and for cluster formation to contain Lm during a secondary oral infection. In addition, we observed the induction of CXCL9 in areas surrounding the clusters, which has been shown to mediate rapid mobilization of memory αβ T cells to prevent further spread of bacterial infection. We believe these observations support an exciting new role for memory gamma delta T cells in the containment of intestinal pathogens.
Proliferation, cytokine secretion, and immunosuppressive activity of YFAK-specific regulatory T cells. (THER3P.885)
Authors: Ng Sze-Ling , Strominger Jack
Published: January 2023

Autoimmune diseases occur when there is misregulation within the immune system. Regulatory T cells (Tregs) are critical for the regulation of this immune response, however, little is known about their own regulation. The induction and activation of Tregs have been shown to alleviate symptoms of autoimmune disease such as human multiple sclerosis (MS). Our lab has identified Tregs that are activated by the random amino acid copolymer YFAK. These YFAK-specific Tregs can reduce the symptoms of experimental autoimmune encephalomyelitis (EAE), the animal model of MS, more effectively than YEAK (Copaxone), a drug currently used in MS therapy. We have generated both YFAK-specific T cell lines as well as retrogenic mice expressing YFAK-specific T cell receptors (TCR) in order to study the regulation and activation of these beneficial Tregs. We find that these YFAK-specific Tregs will proliferate upon stimulation with glucococorticoid-induced TNF receptor ligand (GITRL). In addition, these Tregs can be activated to produce high levels of anti-inflammatory cytokines, and are immunosuppressive against conventional T cells. We plan to further characterize these YFAK-specific Tregs, as well as to utilize the TCR retrogenic mice in in vivo functional experiments. These studies will help to further explore the use of YFAK-specific Tregs in the prevention of autoimmune diseases such as multiple sclerosis.
GM-CSF-neuroantigen fusion proteins inhibit experimental autoimmune encephalomyelitis upon injection into lymphatics primed by encephalitogenic peptide and complete Freund’s adjuvant. (THER3P.878)
Authors: Curtis Alan , Islam Touhidul , Wilkinson Daniel , Mannie Mark
Published: January 2023

Single-chain fusion proteins comprised of GM-CSF and a myelin neuroantigen (NAg) epitope are potent, NAg-specific inhibitors of rat and mouse models of experimental autoimmune encephalomyelitis (EAE). This study provided evidence that GMCSF-MOG (myelin oligodendrocyte glycoprotein 35-55 peptide) inhibited EAE when co-injected adjacent to the encephalitogenic MOG35-55/ complete Freund’s adjuvant (CFA) emulsion. Furthermore, GMCSF-MOG had tolerogenic activity in the C57BL/6 model of chronic EAE when directly incorporated with MOG35-55 in the CFA emulsion. Likewise, GMCSF-PLP (proteolipid protein 139-151 peptide) inhibited relapsing-remitting EAE in SJL mice when mixed with PLP139-151 in the CFA emulsion. In vitro studies revealed that GMCSF-MOG stimulated the growth and differentiation of inflammatory dendritic cells (DC) and targeted the MOG35-55 domain for potentiated antigen presentation. However, presentation of MOG35-55 by these inflammatory DC was inhibitory, and the inhibitory mechanism in part depended upon the elaboration of nitric oxide. In conclusion, GMCSF-NAg was tolerogenic in CFA-primed lymphatics by a mechanism associated with inflammatory DC, targeted NAg presentation, and nitric oxide production. The inhibitory activity of GMCSF-NAg in CFA-primed lymphatics distinguished GMCSF-NAg fusion proteins as inflammation-dependent tolerogens mechanistically distinct from peptide or protein-based tolerogens.
Effects of hormone receptor activation on tumor associated dendritic cell signaling (TUM4P.921)
Authors: Watkins Stephanie , Thompson Matthew
Published: January 2023

Gender disparity has been observed in cancer in relation to incidence, disease aggressiveness and prognosis. However, the role of the immune system and the activation of an anti-tumor immune response based on gender is unclear. Some of the most successful cancer therapies to date have focused on hormone receptor blockade in tumor cells. Immune suppression is a major hurdle to overcome in the effort to find an effective and durable treatment against cancer. The underlying causes of immune suppression are complex and not completely understood. Hormones, including androgen and estradiol, are also known to dampen immune responses, but immune suppression or activation has not been studied during hormone deprivation therapy. Many studies indicate that tumor-associated dendritic cells within the tumor microenvironment are key regulators of cytotoxic T cell function and may serve as a critical target for immunotherapy. Therefore, in the current study, we demonstrate that hormone receptor signaling, androgen receptor and estrogen receptor in dendritic cells has a critical impact on the transcriptional regulation of factors known to induce immune suppression and may be a key factor to consider when designing immune therapies. Furthermore, the data may also implicate the patient’s gender as an important consideration in boosting anti-tumor immune responses.
Immunostimulatory early phenotype of tumor-associated macrophages does not predict tumor growth outcome in the HLA-DR mouse model of prostate cancer (TUM4P.923)
Authors: Klyushnenkova Elena , Kim David , Alexander Richard , Riabov Vladimir
Published: January 2023

Tumor-associated macrophages (TAM) were shown to support progression of many solid tumors. However, anti-tumor properties of TAM were also reported in several types of cancer. Here, we investigated the phenotype and functions of TAM in two transgenic mouse models of prostate cancer displaying striking differences in the tumor growth outcome. Mice expressing Prostate-Specific Antigen as a self-antigen in the prostate (PSA tg mice) rejected PSA-expressing TRAMP tumors with high frequency. However, introduction of HLA-DRB1*1501(DR2b) transgene aggravated the growth of TRAMP-PSA tumors in DR2bxPSA F1 mice. Despite the difference in the tumor growth outcome, tumors in both strains were equally and intensively infiltrated by TAM at the first week after tumor challenge. TAM exhibited mixed M1/M2 polarization characterized by the production of pro-inflammatory (TNFα, IL1β) and anti-inflammatory (IL10) cytokines. They also secreted T cell/NK cell-recruiting chemokines (CXCL10, CCL2, CCL3, CCL4, CCL5) and expressed high levels of MHC class II and co-stimulatory molecules. Functionally, TAM demonstrated pronounced immunostimulatory activity in a mixed leukocyte reaction and induced apoptosis of tumor cells. We conclude that during early stages of tumor progression, the phenotype and functional properties of TAM did not predict tumor growth outcome in our model. Furthermore, we demonstrate that at this stage TAM have a potential to activate T cell immunity and mediate anti-tumor effects.
MicroRNA-9 modulates the maturation and function of myeloid-derived suppressor cells by targeting Runx1 (TUM4P.902)
Authors: Tian Jie , Rui Ke , Zhang Yue , Tian Xinyu , Wang Shengjun
Published: January 2023

Myeloid-derived suppressor cells (MDSCs) accumulate in tumor-bearing hosts and play a major role in tumor-induced immunosuppression, which hampers effective immunotherapeutic approaches. Our previous study showed that β-glucans could differentiate monocytic MDSCs (M-MDSCs) into mature myeloid cells and impair the suppressive function of MDSCs. However, the molecular networks are largely unknown. MicroRNAs (miRNAs) are involved in the regulation of immunity, including the immune cell development and differentiation. In this study, we identified that the expression of microRNA-9 (miR-9) was dramatically reduced after β-glucan stimulation, which was considered an essential player in modulating the maturation and function of MDSCs. Overexpressing miR-9 could significantly enhance the activity of MDSCs and reverse β-glucan induced maturation by targeting runt related transcription factor 1 (Runx1) while down-regulating miR-9 remarkably enhanced the maturation of MDSCs and impaired the suppressive effect of the cells. In addition, down-regulation of Runx1 resulted in increased suppressive effect of MDSCs and inhibited the maturation process. Furthermore, knockdown of miR-9 significantly impaired the activity of MDSCs and inhibited the tumor growth in vivo. Collectively, our findings reveal that miR-9 expression in MDSCs plays a critical role in the molecular events governing the maturation and function of MDSCs and might be identified as a potential target in cancer therapy.
Particulate beta-glucan modulates myeloid-derived suppressor cells (MDSC) activity in tumor-bearing mice and lung cancer patients (TUM4P.931)
Authors: Albeituni Sabrin , Ding Chuanlin , Liu Min , Hu Xiaoling , Luo Fengling , Yan Jun
Published: January 2023

Whole glucan particles (WGP) are microparticles of 1,3-beta glucan with 1,6-branches purified from the yeast Saccharamyces cerevisiae. Previous studies have reported its therapeutic efficacy in different tumor models through the activation of dendritic cells and macrophages via the c-type lectin receptor dectin-1. In this study, we investigated the effect of WGP treatment on myeloid-derived suppressor cells (MDSC) in mouse tumor models and in patients with non-small cell lung cancer (NSCLC). MDSC are a heterogeneous population of immature myeloid cells with suppressive activity that accumulate in cancer. Here we found that tumor-bearing mice treated orally with WGP have a significantly lower tumor weight with a significant decrease in the percentage of MDSC in the spleen compared to untreated animals in vivo. Patients with NSCLC treated with WGP prior to surgery have a decreased trend in MDSC percentage and increased respiratory burst of neutrophils in the peripheral blood. In addition, in vitro stimulation of MDSC subsets with WGP abrogates STAT3 phosphorylation in polymorphonuclear MDSC and enhances STAT3 phosphorylation in monocytic MDSC, with the activation of Erk1/2 phosphorylation in both subsets, suggesting that WGP differently modulates MDSC subsets. Moreover, MDSC treated with WGP have a decreased suppressive activity when cocultured with OVA-specific OTI and OTII splenocytes. Overall, these data indicate that WGP might be a new modulator of MDSC function in cancer.
ER stress response regulates the fate of myeloid-derived suppressor cells through TRAIL receptors mediated apoptosis (TUM4P.907)
Authors: Condamine Thomas , Kumar Vinit , Ramachandran Indu , Youn Je-In , Celis Esteban , Finnberg Niklas , El-Deiry Wafik , Winograd Rafael , Vonderheide Robert , English Nickolas , Knight Stella , Yagita Hideo , Mccaffrey Judith , Antonia Scott , Hockstein Neil , Witt Robert , Masters Gregory , Bauer Thomas , Gabrilovich Dmitry
Published: January 2023

We studied the fate of myeloid-derived suppressor cells (MDSC) in cancer. Unexpectedly, MDSC had lower viability and a shorter half-life than their control counterparts neutrophils and monocytes. This effect was due to increased apoptosis mediated by the changes in TRAIL receptors (TRAIL-R) expression. Targeting TRAIL-R did not affect myeloid cells from naïve mice, but dramatically reduced the presence of MDSC and improved the immune responses in tumor-bearing mice. Pro-inflammatory cytokines did not affect TRAIL-R expression. However, induction of ER stress recapitulated changes in TRAIL-R expression observed in tumor-bearing hosts. ER stress response was detected in MDSC isolated from cancer patients and tumor-bearing mice, but not in control neutrophils or monocytes. Block of ER stress abrogated changes in TRAIL-R. Thus, MDSC pathophysiology is linked to ER stress, which shortens their lifespan in the periphery and promotes their expansion in bone marrow. In conclusion, TRAIL-R can be considered for selective targeting of MDSC.
Vaccine self-assembling immune matrix (VacSIM) is a non-viral delivery platform that augments responses to recombinant protein vaccines (VAC6P.940)
Authors: Shollenberger Lisa , Grenfell Rafaella , Samli E. Farah , Harn Donald
Published: January 2023

Vaccination remains the most effective public health tool to prevent infectious disease. However, many vaccines remain marginally effective, especially for immune-compromised populations. To enhance vaccine immunogenicity, we exploited the biphasic property of certain synthetic oligopeptides to create a new vaccine delivery method, VacSIM (vaccine self-assembling immune matrix). Vaccine components are easily mixed with the VacSIM solution for injection, after which the peptides self-assemble into hydrated nanofiber gel-matrices, forming a vaccine depot. Thus, we have a non-viral, inert, biodegradable delivery system, not requiring formulation, which we can use to deliver a multitude of vaccines. Analogous to a treat-dispensing dog toy, VacSIM is a carrier for vaccine components, allowing for gradual release, rather than immediate administration. We believe this depot attracts antigen presenting cells, driving enhanced vaccine-specific responses that improve both the quantity and quality of the response. We have tested this delivery platform with live bacterial vectors, inactivated virus and multiple recombinant protein vaccines. Shown here, VacSIM augments vaccine responses to the recombinant Hepatitis B surface antigen and recombinant HIV Envelope in 2 strains of mice. Enhancement of vaccine responses by VacSIM administration could represent a fundamental paradigm shift in how vaccines are delivered.
Differential contribution of variable heavy and variable light chain domains in viral epitope recognition and neutralization function (VAC6P.950)
Authors: Pasman Yfke , Nagy Eva , Kaushik Azad
Published: January 2023

Antigen (Ag) may be recognized by an immunoglobulin via heavy chain variable domain (VH) alone, light chain variable domain (VL) alone and VH+VL pair. Little is known about relative contribution of VH and/or VL in Ag recognition in the context of virus neutralization function. To understand role of VH and VL, we examined Ag recognition and virus neutralization function of VH, VL and VH+VL of a neutralizing Ab against bovine herpesvirus-1 (BoHV-1), an important cattle pathogen. Monomeric scFv, VH and VL were expressed in Pichia pastoris and purified. The VH alone recognized BoHV-1 in ELISA but VL did not. This is consistent with previous studies that suggested significant role of variable heavy domain in Ag recognition. However, VH alone did not neutralize BoHV-1 in vitro but monomeric scFv, where VH and VL are linked via 18 amino acids, neutralized the virus. Such a divergence in VH and VL effector functions from Ag recognition to virus neutralization reflects fine structural complexities relevant to Ab functions. The VH alone though capable of recognizing the target neutralizing epitope is unable to neutralize BoHV-1 per se. These observations provide novel insight into non-Ag binding functional role of VL where it provides structural and configurational support to VH for accessing the neutralizing viral epitope. To conclude, such subtle differences in VH and VL contributions to Ab effector functions deserve consideration while designing Ab-based anti-viral therapeutics.