bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of a novel tool for Francisella tularensis immunology (MUC1P.915)
Authors: Place David , Kirimanjeswara Girish
Published: January 2023

Tularemia, an acute infection caused by Francisella tularensis, can manifest in various forms. Pulmonary tularemia developed after an aerosol exposure to the bacterium has a high risk of mortality in unvaccinated individuals. Therefore, development of a vaccine is of critcal importance but is hampered by a relative lack of understanding of the protective adaptive immune response. Absence of immunological tools to study protective T cell responses has been one of the key hurdles in establishing the profile of immune responses to F. tularensis. We have developed a novel F. tularensis LVS, which constitutively expresses a vgrG-OVA fusion protein containing the epitopes OVA257-264 and OVA323-339. This will allow researchers to study antigen-specifc T cell responses during infecton by F. tularensis. Robust expression of this construct was confirmed by immunoblot from bacterial lysates. An additional chromosomally-integrated expression construct is also being generated for more stable expression throughout in vivo infections. This new tool will allow us to study the memory response to Francisella vaccinaton in ways previously difficult to do. Inital experiments are underway using OT-I and OT-II transgenic mice to study the pathogen-specifc response to Francisella following vaccinaton by the intradermal and intranasal routes in order to determine the ideal conditons for improving survival after pulmonary challenge, which remains a concern in a potental bioterror atack.
In situ imaging unveiled location of protective endogenous CD8 T cell response in lung (MUC1P.916)
Authors: Gilchuk Pavlo , Guy Clifford , Hill Timothy , Rabacal Whitney , Sebzda Eric , Green Douglas , Joyce Sebastian
Published: January 2023

The anatomic distribution of protective endogenous memory CD8 T cell response in the lung—a common site of pathogen invasion—is poorly understood. We report that endogenous CD8 T cell memory installed by local pulmonary, but not systemic, vaccination resulted in faster viral control and enhanced protection to a lethal respiratory viral challenge in mice. Enhanced protection depended on elicitation of a distinct CXCR3LO resident CD8 T cell memory population (CD8 TRM) confined to the pulmonary interstitium. CD8 TRM sensed the pathogen and rapidly initiated a local immune response before inflammation-induced recruitment of lung vascular and/or circulating memory CD8 cells. Imaging of protective CD8 T cell response in lungs by exploiting in situ staining with fluorescent MHC class I tetramers coupled with Sheet Plane Illumination Microscopy (SPIM) platform uncovered that CD8 TRM are retained within immunologically correct locations, which include the delicate alveoli walls outside of the alveolar capillary network - the most vulnerable sites affected by lower airway infection. Such a location of memory CD8 T cell response may allow prompt surveillance of infected alveolar epithelium without need for the egress into the alveolar space. In sum, our study unveiled novel dimensional information of memory T cell response that localize to those anatomic sites of the lung that are critical for host defense.
Trace levels of staphylococcal enterotoxin bioactivity are concealed in a mucosal niche during pulmonary inflammation (MUC1P.917)
Authors: Menoret Antoine , Svedova Julia , Behl Bharat , Vella Anthony
Published: January 2023

Pathogen and cellular by-products released during infection or trauma are critical for initiating mucosal inflammation. This was studied in mice undergoing pulmonary inflammation after Staphylococcal enterotoxin A (SEA) inhalation. A potent pro-inflammatory factor was detected using a sensitive bioassay suggesting the presence of an alarmin, a danger-associated molecular pattern (DAMP), and/or a cytokine. Highly purified bronchoalveolar lavage fluid (BALF) fractions obtained by sequential chromatography were screened for bioactivity and subjected to mass spectrometry. SEA was the only identified protein with known inflammatory potential, and unexpectedly, it co-purified with immunosuppressive proteins. Among them was lactoferrin, which inhibited SEA and anti-CD3/-CD28 stimulation by promoting T cell death and reducing TNF synthesis. Higher doses of lactoferrin were required to inhibit effector compared to resting T cells. This inhibition relied on the continual presence of lactoferrin rather than a programming event. The data show that bioactive SEA is constituted in a mucosal niche within BALF even after the initiation of inflammation. These results may have clinical value in human diagnostic settings since traces levels of SEA can be detected using a sensitive bioassay, and may help pinpoint potential mediators of lung inflammation when molecular approaches fail. Submitted to American Journal of Respiratory Cell and Molecular Biology.
Human HLA-G+ extravillous trophoblasts: immune activating cells that interact with decidual leucocytes (MUC2P.921)
Authors: Tilburgs Tamara , Crespo Angela , van der Zwan Anita , Moffett Ashley , Strominger Jack
Published: January 2023

Invading HLA-G+ extravillous trophoblasts (EVT) are believed to play a key role in the prevention of a maternal immune attack on foreign fetal tissues. EVT are difficult to study due to their low frequency and lack of proliferative capacity. Here highly purified HLA-G+ EVT and HLA-G- villous trophoblast (VT) were isolated. Culture on fibronectin increased HLA-G expression on EVT but differentiation from VT into EVT was not observed. Moreover microarray analysis demonstrated that VT and EVT have more than 4200 differentially expressed genes. Functional gene set enrichment analysis (GSEA) revealed a striking immune activating potential for EVT that is absent in VT. Co-culture of HLA-G+ EVT with sample matched decidual NK, macrophages, CD4+ and CD8+ T cells were established and demonstrated interactions of all leukocyte types with EVT. Interaction of EVT with CD4+ T cells resulted in increased proportion of CD4+CD25hiFOXP3+ Tregs and increased FOXP3 protein level in these cells. However, EVT did not enhance cytokine secretion in dNK whereas stimulation of dNK using mitogens or classical NK targets confirmed the distinct cytokine profiles of dNK and pNK. Thus EVT are specialized cells with an immune activating profile whose properties are not imitated by HLA-G expressing cell lines. Careful validation of EVT-leukocyte interactions using primary HLA-G+ EVT needs to be carried out to understand the unique contribution of EVT to the decidual immune response in human pregnancy.
Increased expression of antimicrobial peptides in the cervix during Trichomonas vaginalis infection (MUC2P.923)
Authors: Makinde Hadijat , Novak Richard , Landay Alan , Spear Gregory
Published: January 2023

The lower genital tract is a unique mucosal environment and further studies are needed to define the types of immune responses that occur during genital infections. We investigated the expression of antimicrobial proteins/peptides (AMP) and cytokines produced in response to Trichomonas vaginalis (TV) infection. A cohort of women (30 TV+, 30 TV-) aged 18-45 that was at high risk of acquiring HIV was studied. mRNA was isolated from cervical cytobrush samples, reverse transcribed, and real time PCR used to determine AMP and cytokine expression. Cytokines were measured by ELISA in cervical vaginal lavage (CVL). TV+ samples had significantly higher mRNA levels of S100A7, S100A8, S100A9 and Muc1 than TV- samples. In contrast, lactoferrin was lower in the TV+ group. For defensins (HBDs), mRNA for HBD4 was significantly higher in TV+ samples, while HBD103 was lower in TV+ samples, but there was no difference in HBD1. Cathelicidin was not different between TV+ and TV- samples. mRNA for RegIIIγ was detected in only four samples. Although a role in inducing expression of AMPs has been shown for IL-22 and IL-17A, these two cytokines, as well as IFNγ, were not different in TV+ and TV- groups. However, IL-17 protein, but not IL-22 was higher in TV+ CVL samples (p < 0.05). Our data identify AMPs that have altered expression in the cervix during TV infection suggesting that these AMPs might have an important role in immunity against TV infection.
Role of NK cells in IFN-epsilon-mediated protection against female reproductive tract infection (MUC2P.925)
Authors: Hansbro Philip , Mayall Jemma , Mangan Niamh , Starkey Malcolm , Kim Richard , Hertzog Paul , Horvat Jay
Published: January 2023

Chlamydia trachomatis is the most common bacterial STI and frequently results in reproductive tract (RT) sequelae such as pelvic inflammatory disease and infertility. However, the immune processes involved in the clearance and immunopathology of Chlamydia infection are not well understood. In previous studies we showed that IFN-ε, a novel type I IFN that is exclusively and constitutively expressed in the female RT, plays an important role in protecting against Chlamydia infections. Here, we examined the effects of IFN-ɛ on cellular responses in the female RT in order to elucidate the mechanisms by which IFN-ɛ protects against Chlamydia infections. Female WT and IFN-ε-/- C57BL/6 mice were pre-treated with progesterone and infected intra-vaginally with Chlamydia muridarum or sham-infected. Uterine horns were harvested and the effects of IFN-ε deficiency on Chlamydia infection, immune factor expression and cellular infiltration were assessed using real-time qPCR and flow cytometry. We show that IFN-ε-/- mice have increased Chlamydia 16S expression in the upper RT which correlated with fewer NK cells and tissue-resident uterine NK cells at 3 days post infection. IFN-γ+ CD45+ cells were also decreased in the infected IFN-ε-/- mice, of which over 60% were NK cells. These changes were associated with reduced iNOS and STAT1 expression. These findings suggest that IFN-ε may protect against Chlamydia RT infections by potentiating the recruitment of protective IFN-γ-producing NK cells.
Impacts of ethanol and nicotine withdrawal on fecal immunoglobulin A levels in rats (MUC3P.947)
Authors: Harris Henry , Lingg Ryan , Hayes Dayna , Redmond Sarah
Published: January 2023

Alcohol and nicotine are both well known to cause depression of immune response; however, the effects of withdrawal from these substances have not been extensively explored. The impacts of exposure may arise from direct depression of the immune system or from the stress associated with exposure and withdrawal. In this study the immune response of adult male Rattus norvegicus was tested following exposure to both alcohol and nicotine, alcohol alone, nicotine alone, or neither (n=4/group). Nicotine or saline was administered 3 times per day for 10 days (0.3 mg/kg in saline; subcutaneous). Alcohol (25%w/v in nutritionally complete diet; intragastric) or isocaloric dextrose-containing diet was administered during days 7-10 of nicotine administration following the same thrice-daily schedule. Fecal samples were collected 4, 7, 11 and 14 days post exposure and fecal extractions were tested via ELISA to measure immunoglobulin A (IgA) levels. The effects of ethanol exposure, nicotine exposure, time post-exposure, and the interactions of the three were tested using a 3-way ANOVA. There was a significant decrease in IgA levels across groups on day 7 (p = 0.015), followed by an increase in IgA levels measured on day 10 in ethanol exposed groups (p = 0.011). This fluctuation in mucosal immunity following binge exposure to ethanol suggests that the impacts of even short term alcohol use impact the immune response during a withdrawal period.
Maximal use systemic exposure study evaluating AN2728, a novel boron-based small molecule, for the treatment of pediatric and adolescent subjects with mild-to-moderate atopic dermatitis (THER2P.959)
Authors: Kircik Leon , Call Robert , Tschen Eduardo , Draelos Zoe , Van Syoc Merrie , Zane Lee , Hebert Adelaide
Published: January 2023

AN2728, a novel boron-based compound, inhibits phosphodiesterase-4 and reduces proinflammatory cytokine production possibly associated with atopic dermatitis(AD). This open-label maximal-use study evaluated systemic exposure, PK, and safety of AN2728 Ointment, 2% BID for 28 days in children and adolescents with mild-to-moderate AD(N=34; 3 cohorts based on age and minimum % treatable body surface area [%BSA] affected: 2-5y [≥35%]; 6-11y [≥35%]; 12-17y [≥25%]). Disease severity was measured using Investigator’s Static Global Assessment (ISGA; 0, clear to 4, severe), signs/symptoms score (0, none to 3, severe) and %BSA affected. At day 29, 65% of subjects had ISGA scores of clear or almost clear and 47% had scores of clear or almost clear and a ≥2-grade improvement vs baseline. Marked reductions vs baseline were seen for AD signs and symptoms (pruritus, erythema, lichenification, excoriation, exudation) throughout treatment. Mean pruritus scores improved by ~60% vs baseline as early as treatment day 5. Mean %BSA affected decreased by 78% after 4 weeks of treatment. The most common treatment-related adverse events were application site reactions (n=12) which generally were mild or moderate and resolved spontaneously. One patient withdrew due to application site pain. PK results showed low AN2728 blood levels similar to those previously seen in adults after adjusting for %BSA treated. AN2728 Ointment, 2% may be safe and effective in subjects aged ≥2 years with mild-to-moderate AD.
Adoptive cellular gene therapy for the treatment of experimental autoimmune polychondritis ear disease (THER2P.966)
Authors: Zhou Bin , Liao Yonggan , Guo Yunkai , H Tarner Ingo , Liao Chunfen , Chen Sisi , Habiby Kermany Mohammad , Tu Hanjun , Zhong Sen
Published: January 2023

Clinical therapy of autoimmune diseases used to be limited to non-specific immune-suppressive agents and was, for the most part, based on chance observations. Currently, gene therapy research aims at achieving higher specificity and thus less adverse side effects. This concept comprises the adoptive transfer of autologous T-cells that have been retrovirally transduced ex vivo to express and deliver immune-regulatory gene products to sites of autoimmune inflammation. In the animal model of collagen-induced autoimmune polychondritis ear disease (CIAPED), the adoptive transfer of IL-12p40 expressing collagen type II (CII)-specific CD4+ T-cell hybridomas resulted in a significantly lower disease incidence and severity compared with untreated or vector-only treated animals. In vivo cell detection using bioluminescent labels showed that transferred CII-reactive T-cell hybridomas accumulated in inflamed ear lobes in mice with CIAPED. In vitro analysis demonstrated that IL-12p40-transduced T-cells did not affect the antigen-specific T-cell activation or systemic anti-CII antibody responses. However, IL-12p40-transduced T-cells suppressed IFN-γ and augmented IL-4 production, indicating its potential to act therapeutically by interrupting Th1-mediated inflammatory responses via augmenting Th2 responses. These results indicate that the local delivery of IL-12p40 by T-cells could inhibit CIAPED by suppressing autoimmune responses at the site of inflammation.
Neuroprotective effects of bee venom phospholipase A2 mediated by the suppression of neuroinflammatory responses in the 3xTg AD mouse model of Alzheimer’s disease (THER2P.967)
Authors: Bae Hyunsu , Ye Minsook , Lee Chanju , Chung Hwan-Suck , Shim Insop
Published: January 2023

Alzheimer’s disease (AD) is a severe neuroinflammatory disease. CD4+Foxp3+ regulatory T cells (Tregs) play a pivotal role in maintaining immune tolerance in various inflammatory diseases. In this study, we sought to determine whether bee venom PLA2 promotes cognitive function in and modulates the patho-immunological features of AD in 3xTg-AD mice. PLA2 treatment significantly enhanced the cognitive function of the 3xTg-AD mice and increased glucose metabolism, as assessed with 18F-2 fluoro-2-deoxy-D-glucose ([F-18] FDG) positron emission tomography (PET). The levels of amyloid beta (Aβ) deposits in the hippocampus were dramatically decreased following PLA2 treatment. This neuro-protective effect of PLA2 was associated with microglial deactivation and a reduction in CD4+ T cell infiltration. Interestingly, the neuroprotective effects of PLA2 were abolished in Treg-depleted mice. Therefore, the present studies strongly suggest that the modulation of peripheral immune tolerance by Treg might contribute to the neuroprotective effect of PLA2 in the 3xTg AD mice.
Negative regulation of pathogenic TH17 cells by IK cytokine ameliorates inflammatory arthritis (THER3P.969)
Authors: Park Hye-Lim , Min Jun-Ki , Lee Sang-Myeong , Moon Su-Jin , Lee Dong-Hee , Nam Jae-Hwan
Published: January 2023

The pathogenesis of inflammatory autoimmune rheumatoid arthritis (RA) is driven by the activation of macrophage, and the development of pathogenic interferon-γ (IFN-γ)- producing and interleukin-17 (IL-17)- producing CD4+ T cells (TH1 and TH17). Here we showed that truncated IK (tIK) cytokine, inhibitor of IFN-γ induced MHC class II expression, ameliorated RA in a mouse model, which was associated with decreases in joint swelling and bone destruction, as well as the incidence. However, the detailed mechanism of tIK-reduced inflammatory arthritis is still unclear. Of note, we found that the splenocytes of tIK-Tg after LPS treatment showed the induction of A20 (also termed as TNFAIP3) compared with those of WT in microarray analysis. We also found that differentiation of CD4+ T cells from tIK-Tg to TH17 cells was decreased. Consistent with this, A20 were highly expressed in CD4+ T cells from tIK-Tg under TH17 cells polarizing condition, which was associated with reduced NF-κB and p38 signaling pathways. Thus, these findings suggest therapeutic and regulatory function for IK cytokine, supporting the strategy for drugs to target pathogenic TH17 cells in inflammatory arthritis.
IL-12p40 homodimer (p40)2 ameliorates experimental autoimmune arthritis (THER3P.975)
Authors: Lee Seon-Young , Jung Young Ok , Kim Ho-Youn , Kim Doo-Jin , Sung YoungChul , Cho Mi-La
Published: January 2023

Interleukin (IL)-23 is the key cytokine that induces the expansion of helper T (Th) 17 cells. It is composed of p19 and p40 subunits of IL-12. The p40 subunit binds competitively to the receptor of IL-23 and blocks its activity. Our aim was to assess the preventive and therapeutic effect of the (p40)2 subunit in autoimmune arthritis animal models. In the present study, by using IL-1receptor antagonist knockout mice (IL-1RaKO) and a collagen-induced arthritis (CIA) model, we inspected the suppressive effect of the IL-12p40 homodimer ((p40)2) on inflammatory arthritis. We demonstrated that the recombinant adenovirus expressing mouse (p40)2 (rAd/(p40)2) model prevented the development of arthritis when given before the onset of arthritis. It also decreased the arthritis index and joint erosions after it was transferred once arthritis was established in the mouse model. (p40)2 inhibited the production of inflammatory cytokines and antigen-specific T cell proliferation. It also induced the CD4+CD25+Foxp3 regulatory T (Treg) cells in vitro and in vivo, whereas the generation of RORgt and Th17 cells was suppressed. The induction process of Treg cells and the suppression of Th17 cells were mediated via the activated STAT5 and suppressed STAT3. Conclusion: Our data suggested that (p40)2 suppressed inflammatory arthritis successfully. This could be a useful therapeutic approach in autoimmune arthritis to regulate the Th17/Treg balance and IL-23 signaling.
PD-L1 blockade and upregulation in an ovarian cancer treatment model (TUM2P.1017)
Authors: Grabosch Shannon , Zhang Lixin , Zeng Feitianzhi , Mony Jyothi , Ma Tianzhou , Tseng George , Edwards Robert , Vlad Anda
Published: January 2023

Objective: We explored mechanisms of PD-L1 upregulation in ovarian cancer cells and tested in vivo the efficacy of intraperitoneal anti-PD-L1 therapy in two different ovarian cancer mouse models. Methods: We have derived several novel murine ovarian cancer cell lines from orthotopic ovarian tumors of triple transgenic mice. 129x SvJ and C57Bl/6 tumor-bearing mice were treated with 200 μg anti-PD-L1 antibody (or rat IgG as control) every two weeks for three doses. Immune gene profiling of splenocytes collected at necropsy was performed using 561 Nanostring probes. Results: Cell cycle analysis of in vitro cultured tumor cells demonstrates that PD-L1 expression increases preferentially on cells in G2/M cells. Exposure to cisplatin modifies PD-L1 levels, although the effect varies according to tumors’ baseline susceptibility. In vivo, we observed murine PD-L1 upregulation on ascites resident tumor cells, an effect reproduced ex vivo upon exposure of tumor cells to ascites fluid. PD-L1 blockade triggers increase in survival in both preclinical models with immune gene signatures pointing to cytotoxic anti-tumor immune response. Conclusion: PD-L1 expression increases in response to inflammatory stimuli from cell-free ascites and shows preference for dividing cells. PD-L1 upregulation varies according to tumor cells’ susceptibility to cisplatin. PD-L1 blockade increases systemic T cell responses and intratumoral T cell accumulation suggesting therapeutic potential in ovarian cancer.
Immunization of CD200R1KO mice post surgical resection of EMT6 breast cancer leads to long-term (>1year) cure of metastatic tumor growth (TUM2P.1022)
Authors: Gorczynski R. , Khatri Ismat , Podnos Anna
Published: January 2023

Attenuated expression of CD200 decreases local tumor growth and metastasis in immunocompetent animals. We explored the ability to cure BALB/c CD200KO or CD200R1KO mice of tumors ≤1cm3 in size by surgical resection of localized tumor, followed by immunization with irradiated EMT6 cells using Monophosphoryl Lipid A (MPLA:25υg/mouse) as adjuvant. Controls developed pulmonary and liver metastases within 30 days of surgery. Protection was seen in both CD200KO or CD200R1KO mice, with no macroscopic lung/liver metastases observed in CD200R1KO mice at day 300. After resection and immunization, draining lymph nodes from controls contained tumor cells cloned at limiting dilution in vitro before pulmonary and hepatic metastasis was seen. Within the limits of detection of the assay used (sensitivity ~1 in 107 cells) no tumor cells were detected in CD200R1KO mice, and reductions were seen in CD200KO mice. Infusion of anti-CD8 mAb into surgically treated and immunized CD200R1KO mice attenuated protection from both macroscopic (liver/lung) and microscopic (assayed by limiting dilution of DLN) metastasis. Adoptive transfer of lymphocytes from treated CD200R1KO mice to surgically-treated control mice, particularly after Treg depletion attenuated metastatic growth of tumor, an effect abolished by pretreatment with anti-CD8 mAb. We suggest CD200:CD200R attenuates a potentially tumor-protective CD8 host response to breast cancer following immunotherapy using MPLA as adjuvant.
HHLA2 and TMIGD2: A new pathway of the B7/CD28 families and novel targets for human cancer immunotherapy (TUM2P.1028)
Authors: Zang Xingxing
Published: January 2023

We have recently identified HHLA2 as a new member of the B7 family that shares 23-33% similarity to other human B7 proteins and phylogenetically forms a subfamily with B7x and B7-H3. HHLA2 is expressed in humans but not in mice. HHLA2 protein is constitutively expressed on monocytes and induced on B cells, and inhibits proliferation and cytokine production of human CD4 and CD8 T cells in the presence of TCR signaling. HHLA2 protein is not detected in most organs, but is widely expressed in human cancers from the breast, lung, thyroid, melanoma, pancreas, ovary, liver, bladder, colon, prostate, kidney, and esophagus. 56% of patients with triple negative breast cancer have aberrant expression of HHLA2 on their tumors, and high HHLA2 expression is significantly associated with regional lymph node metastasis and stage. HHLA2 copy-number gains are present in 29% of basal breast cancers. HHLA2 binds putative receptors expressed not only on resting and activated CD4 and CD8 T cells but also on APCs. Furthermore, we have identified TMIGD2 as one of the receptors for HHLA2. TMIGD2 shares 23-29% similarity to other human CD28 proteins, but is expressed in humans not in mice. Our results identify HHLA2/TMIGD2 as a new pathway of the B7/CD28 families and suggest that the HHLA2 pathway represents a novel immunosuppressive mechanism within the tumor microenvironment and an attractive target for human cancer therapy.
Ibrutinib, a dual Btk/Itk inhibitor, promotes dendritic cell maturation by inducing IFN-α/β. (TUM2P.1037)
Authors: Natarajan Gayathri , Terrazas Cesar , Oghumu Steve , Varikuti Sanjay , Satoskar Abhay
Published: January 2023

Ibrutinib, a Btk and Itk inhibitor, treats hematological malignancies by acting on lymphoid cells. Additionally, its efficacy against myeloid cell-mediated inflammatory diseases suggests that Ibrutinib could target myeloid cells. Previous studies show that Btk is expressed by murine dendritic cells (DCs). Further, Btk deficient DCs are more mature, express higher levels of MHC-II, co-stimulatory molecules and cytokines and thereby enhance CD4+ T cell activation. Hence, we studied the effect of Ibrutinib on DC function by treating murine bone marrow derived DCs with Ibrutinib during their development. We observed that Ibrutinib-treated DCs upregulate the expressions of MHC-II and co-stimulatory molecules such as CD80 and CD40. Further, Ibrutinib-treated DCs differentially regulate cytokine synthesis by producing more IL-10 while reducing IL-12 production. Ibrutinib-treated DCs promote CD4+ T cell proliferation and enhance T cell derived IFN-γ production. We studied the mechanism by which Ibrutinib promotes DC maturation by studying the expression of cytokines that direct DC maturation. We identified that Ibrutinib-treated DC cultures increase the induction of IFN-α and IFN-β which are known to promote DC maturation and IRF-7, a transcription factor which is expressed upon IFN-α/β activation. Hence, Ibrutinib treatment enhances DC maturation in an IFN-α/β dependent manner and should be further explored in the development of DC-based therapeutic vaccines against cancer.
NK cells and CD8+ T cells cooperate to improve therapeutic responses in melanoma treated with CTLA-4 blockade and IL-2 (TUM2P.1039)
Authors: Kohlhapp Frederick , Broucek Joseph , Hughes Tasha , Huelsmann Erica , Zloza Andrew , Kaufman Howard
Published: January 2023

Immunotherapy is a promising approach for the treatment of metastatic melanoma and combination strategies may have potential for improving therapeutic responses. CTLA-4 blockade increases the CD8:Treg ratio in the tumor microenvironment and IL-2 promotes NK and CD8+ T cell function. Thus, we hypothesized combination IL-2 and CTLA-4 blockade would result in improved anti-tumor responses. This hypothesis was confirmed utilizing the poorly immunogenic B16 melanoma model, with CTLA-4 blocking antibody on days 3, 6, and 9 and/or IL-2 twice daily on days 4-8 after challenge, mirroring clinical use of these agents. Combination immunotherapy resulted in delayed tumor growth and prolonged survival compared to either monotherapy. Depletion studies suggested that both CD8+ T and NK cells were required for the therapeutic effects. In this model, combination treatment resulted in fewer tumor-infiltrating CD8+ T and NK cells but more infiltrating Tregs in the tumor microenvironment when compared to monotherapy CTLA-4 blockade. Further, IL-2 therapy was associated with a reduction in the proportion of highly differentiated/exhausted NK cells in the tumor microenvironment. No significant toxicity was observed in the mice. These results support the combination of IL-2 and CTLA-4 blockade in melanoma and a clinical trial based on these data has been initiated.
Generation and characterization of an anti-PD1-resistant lune cancer line (TUM2P.1040)
Authors: Wang Xiaohong , Ye Huiping , Valdecanas David , Welsh James
Published: January 2023

Immunotherapies targeting PD1/PDL1 have shown unprecedented rates of durable clinical responses in patients with melanoma and lung cancer. However, there is a substantial portion of patients who do not respond to these therapies. The goal of this study is to generate an anti-PD1-resistant tumor line and identify mechanisms of resistance, which should provide novel insights to extend the benefit of immunotherapies. A preclinical lung cancer model was generated by inoculation of a murine lung cancer cell line 344SQ (containing p53 and kras mutations) into syngeneic 129SV/ev mice. Anti-mouse PD1 antibodies were administrated at day 4 post tumor inoculation for a total of 5 doses. The anti-PD1 resistant line was successfully generated after sequential in vivo passaging of a nonresponsive tumor followed by anti-PD1 treatment. Upon the anti-PD1 treatment, the parental line showed 70% tumor reduction, whereas the resistant line showed only 12% tumor reduction. PDL1 expression did not alter in the resistant line. Instead, anti-PD1 treatment remarkably increased MHC I expression in the parental line, whereas the resistant line showed significantly lower MHC I and II expression and failed to upregulate MHC I upon anti-PD1 treatment. The parental and resistant lines showed distinct profiles of tumor-infiltrating immune cells and costimulatory molecule expression in vivo. Next we will study if inducing MHC expression through radiation or HDAC inhibitors will overcome anti-PD1 resistance.
Extracorporeal photopheresis promotes IL-1β production (VAC3P.1054)
Authors: Hackstein Holger , Jakobs Christopher , Peric Adriana , Michel Gabriele , Baal Nelli , Bein Gregor , Bruene Bernhard , Hornung Veit , Yakut Erhan
Published: January 2023

Extracorporeal photopheresis (ECP) is a widely used clinical cell-based therapy exhibiting efficacy in heterogenous immune-mediated diseases such as cutaneous T cell lymphoma, graft versus host disease (GVHD) and organ allograft rejection. Despite its documented efficacy in cancer immunotherapy, little is known regarding the induction of immunostimulatory mediators by ECP. Here we show that ECP promotes marked release of the prototypic immunostimulatory cytokine IL-1β. ECP primes IL-1β production and activates IL-1β maturation and release in the context of caspase-1 activation in monocytes and myeloid dendritic cells. Interestingly, IL-1β maturation by ECP was fully intact in murine cells deficient for caspase-1, suggesting the predominance of an inflammasome-independent pathway for ECP-dependent IL-1β maturation. Clinically, patient analysis revealed significantly increased IL-1β production in stimulated leukapheresis concentrates and peripheral blood samples after ECP. Collectively, these results provide evidence for promotion of IL-1β production by ECP and provide new insight into the immunostimulatory capacity of ECP.
Targeting DNA vaccines to dendritic cells using a small peptide to enhance immune response (VAC4P.1110)
Authors: Ye Chunting , Choi Jang Gi , Abraham Sojan , Shankar Premlata , Swamy Manjunath
Published: January 2023

Dendritic cell targeting of antigens greatly enhances immunogenicity. Although there are several reagents (DEC205, Dectin-1, Langerin, Clec9, nanoparticle, etc) to target protein antigens to DCs, there is still no method to target DNA vaccines directly to DCs. Here we show that a small peptide derived from the rabies virus glycoprotein, fused to protamine residues (RVG-P) can target DNA to DCs resulting in enhanced T-cell and humoral responses. Targeted delivery of DNA vaccine encoding the immunodominant Vaccinia virus B8R gene to DCs with RVG-P was able to re-stimulate Vaccinia-specific memory T cells in vitro. Moreover, a single iv injection of B8R gene bound to RVG-P without any adjuvants was able prime a Vaccinia-specific T-cell response that was able to rapidly clear a subsequent Vaccinia virus challenge in mice. Finally, immunization of mice with a DNA vaccine encoding with West Nile virus (WNV) prM and E proteins via RVG-P elicited high titers of WN neutralizing antibodies that protected mice from lethal WNV challenge. Thus, RVG-P provides a tool to target DNA vaccines to DCs to elicit robust T-cell and humoral immune responses.
Mucosal vaccination with an attenuated strain of Mycobacterium tuberculosis induces a strong central memory response and protects non-human primates against lethal challenge (VAC4P.1104)
Authors: Foreman Taylor , Mehra Smriti , Gautam Uma , Rangel-Moreno Javier , Khader Shabaana , Kaushal Deepak
Published: January 2023

There is an urgent need for new vaccine(s) that protect efficiently against Mycobacterium tuberculosis (Mtb), as well as, to better understand the correlates of a protective host response to Mtb infection. Aerosol infection of macaques with the redox-stress deficient mutant, Mtb:Δ-sigH, resulted in the recruitment of significantly more inducible bronchus-associated lymphoid tissue, which is associated with protection from Mtb infection in mice, macaques, and humans. We therefore hypothesized that vaccination with Mtb:Δ-sigH could protect against a lethal Mtb challenge significantly better than a comparable Bacille Calmette-Guerin (BCG) vaccination. A single vaccination of the aerosol delivered, live-attenuated strain completely protected macaques from lethal Mtb challenge. Protection was associated with three-to-four log lower bacterial burden, significantly reduced granulomatous pathology and clinical features of Mtb infection, as well as, a strong T cell central memory response when compared to BCG-vaccinated animals. A higher magnitude, as well as, breadth of polyfunctional T cell responses was detected in the Mtb:Δ-sigH vaccinated animals. This includes an increased recruitment and turnover of central and effector T cells in the lung post vaccination compared to BCG-vaccinated animals. These results point to the protective qualities induced by a live-attenuated mucosal vaccine for protection against lethal Mtb challenge, and its future in TB vaccine initiatives.
Antibody immunotherapy targeting the self-tumor antigen nucleolin (VAC5P.1129)
Authors: Sutkowski Natalie , Sun Wei , Fernandes Daniel
Published: January 2023

Nucleolin is a multifunctional protein found in the nucleus of all cells, and it’s a self-tumor antigen. It is overexpressed on the plasma membrane and cytoplasm of the two most common forms of leukemia acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL). The cell surface expression of nucleolin on CLL and AML cells, compared with its nuclear profile in normal cells, makes nucleolin an attractive self-antigenic target for antibody immunotherapy. Accordingly, we have utilized our patented platform technology (US Patent 8,715,743) to generate a panel of fully human monoclonal antibodies (HuMAbs) that target nucleolin (NCL) and potently induce AML cell death, while having no effect on normal blood cells (PCT/US2010/057046). Preliminary data indicated that AML cells were killed independently of complement or antibody dependent cellular cytotoxic mechanisms, possibly by interfering with nucleolin’s cellular function. This might have significant therapeutic implications, since the HuMAbs may retain activity in immunocompromised leukemia patients. We hypothesize that the anti-NCL HuMAbs have both immune mediated and immune independent antitumor activity, eliciting nucleolin specific killing through both complement and cell mediated cytotoxic mechanisms, and through interfering with the integral cellular function of nucleolin. Thus, autoreactive antibodies targeting this self-tumor antigen might be important in the treatment of leukemia.
Characterization of T cell responses induced by Flu SAM(NP)® and SAM(M1)® vaccines (VAC5P.1124)
Authors: Magini Diletta , Buccato Scilla , Mangiavacchi Simona , Giovani Cinzia , Montomoli Emanuele , Brito Luis , Maione Domenico , Geall Andrew , Brazzoli Michela , Bertholet Sylvie
Published: January 2023

Vaccination remains the most cost-effective way to control Influenza outbreaks. However, current Influenza vaccines do not provide efficacious heterosubtypic immunity. Therefore, the development of a successful universal vaccination strategy is urgently needed. To achieve this goal, one approach is focused on the induction of a T cell based response versus more conserved flu antigens, such as the Nucleoprotein (NP) and the Matrix protein 1 (M1). The aim of this study was to evaluate the immunogenicity and protective efficacy conferred by conserved NP and M1 antigens expressed by a self-amplifying RNA-based vaccine (SAM(NP)® and SAM(M1)®). We demonstrate that SAM(NP)® and SAM(M1)® are immunogenic in Balb/c mice. SAM(NP)® formulation induced IFNγ+ and LAMP1+ NP-specific cytotoxic CD8+ T cells as well as multifunctional NP-specific CD4+ T cells. SAM(M1) ® elicited a robust polyfunctional CD4 T helper 1 response. Moreover, we show that SAM(NP)® and SAM(M1)® formulations, when administered separately or in combination, provided differential levels of protection against a lethal challenge with mouse-adapted A/PR/8/1934 (H1N1) Influenza virus. SAM(NP)® and SAM(M1)® formulation induced T cell immune responses, potent mediator of heterosubtypic immunity, therefore, they are promising antigens in the design of a cross-protective flu vaccine. Since the SAM® vaccine technology offers several advantages , it could potentially speed up the development of universal flu vaccine candidates.
Anatomic redistribution of natural killer cells within secondary lymphoid organs during virus infection (VIR1P.1138)
Authors: Moran Michael , Rydyznski Carolyn , Waggoner Stephen
Published: January 2023

Natural killer (NK) cells are innate lymphocytes that classically function to restrict microbial infection and tumor development. NK cells are also important regulators of adaptive immunity. Specifically, we found that NK cells inhibited antiviral humoral immunity by restricting the germinal center (GC) response via a perforin-dependent cytotoxic mechanism during the initial few days of lymphocytic choriomeningitis virus (LCMV) infection. We hypothesize that this NK cell suppression of antiviral B cells requires redistribution of activated NK cells to regions of secondary lymphoid tissues where T-B cell interactions shape the GC reaction. NK cells were predominately localized within the red pulp of the spleens of uninfected mice, but infection with LCMV triggered a redistribution of NK cells to the white pulp. At day 3 p.i, many NK cells were present in T cell rich areas, most notably at the T-B cell interface where follicular helper T cells (TFH) typically reside. In addition, a small population of NK cells penetrated deep into the B cell follicles. The redistribution of NK cells was transient, with most NK cells returning to the red pulp by day 6 p.i. We speculate that white pulp localization is stimulated by altered expression of chemokine receptors by NK cells, and that this proximity to the GC facilitates direct interactions between NK cells and TFH or GC B cells, thereby contributing to diminished GC responses in the presence of NK cells.
EAE symptoms are enhanced by gammaherpesvirus latenlty infetected memory B cells (VIR1P.1143)
Authors: Marquez Ana , Casiraghi Costanza , Shanina Iryina , Horwitz Marc
Published: January 2023

While the description of the genetic and environmental factors responsible for the onset of Multiple Sclerosis has remained elusive, it is believed that previous infection with Epstein Barr Virus (EBV) plays an important role in the development of MS. Our lab has hypothesized that latent EBV infection alters the immune system’s response in genetically predisposed individuals, thereby exacerbating autoimmunity. The study of this relationship is difficult, however, since EBV only infects humans. To address this question we previously developed a murine model where latent infection with gamma herpesvirus 68 (γHV-68), the murine homolog to EBV, enhances the symptoms of experimental autoimmune encephalomyelitis (EAE), which closely resembles human MS pathogenesis. These enhanced symptoms are driven by the upregulati/on of CD40 on dendritic cells in latently infected mice, which drives the priming of a strong Th1 response as well as a decrease in Treg frequencies. Latency of γHV-68 mainly occurs in memory B cells. Here, we demonstrate that latently infected memory B cells are capable of enhancing EAE symptoms when transferred from mice latently infected with γHV-68 into uninfected mice. To understand the impact of γHV-68 on B cells, we propose a mechanism where type 1 interferons play an important role in skewing a Th1 response in latently infected mice. This is a novel mechanism that describes how EBV latency can spur the onset of MS and potentially other autoimmune diseases.
Greater avidity and phagocytic activity of HIV gp41-specific serum IgA antibodies in elite controllers compared to subjects on antiretroviral therapy (VIR1P.1144)
Authors: Nabi Rafiq , Wei Qing , Moldoveanu Zina , Durkin Helen , Greenblatt Ruth , Herold Betsy , Nowicki Marek , Young Mary , Golub Elizabeth , Landay Alan , Mestecky Jiri , Kozlowski Pamela
Published: January 2023

Identification of the mechanisms of natural control of HIV replication in elite controllers (EC) may inform approaches for prevention of HIV. We investigated whether EC produce envelope-specific serum IgA responses of greater magnitude, avidity and function compared to subjects on combination antiretroviral therapy (cART). Serum was obtained from age- and race-matched HIV-uninfected women and infected women classified as EC or cART controllers (HC) or noncontrollers (HN). The EC and HC had undetectable viremia and CD4 T cell counts > 450/µl. The HN had high viremia and CD4 T cells < 250/µl. Concentrations and avidity of IgA and IgG against Clade B consensus gp120 and gp41BAL proteins were determined using ELISA. THP-1 monocytes and fluorescent beads coated with gp120 or gp41 were used to evaluate phagocytosis in whole serum and in IgA- or IgG-depleted serum. The magnitude of IgA and IgG responses to gp120 and gp41 was not found to differ between groups. Also, no differences were observed for avidity and phagocytic activity of gp120-specific antibodies. However, avidity and phagocytic activity of the anti-gp41 IgA in EC was significantly greater (p = 0.0186 or p = 0.0054 compared to HC or HN; p = 0.0474 compared to HC, respectively). These data indicate that, in EC, affinity maturation of IgA antibodies to gp41 may occur to a greater extent, resulting in gp41-specific IgA with greater antiviral function. Induction of gp41-specific IgA should be considered in vaccine design.
Inhibition of T follicular helper cells mediated immune evasion in Japanese encephalitis virus infection (VIR1P.1146)
Authors: Cui Min , Wang Chong , Yuan Jiaolong , Wang Kunlong
Published: January 2023

Japanese encephalitis virus (JEV) is a mosquito-borne neurotropic virus, which can escape from the peripheral immune response resulting in serious neuroinflammation. However, the mechanism of JEV immune evasion still remains unclear. In our study, it was found that both CD3+ and CD4+ T cell population dramatically decreased in the early stage of infection. And, T Follicular Helper Cell (TFH) counts reduced obviously accompanied with the decline of B cells, following the CD4+CD25+FoxP3+ regulatory T cells (Tregs) increased in the peripheral immune organs and blood after JEV infection. Co-inhibitory molecules CD279 (PD-1) and CD272 (BTLA) were upregulated on CD4+ T cells in JEV infected mouse, while CD274 (PD-1 ligand 1) was also upregulated on dendritic cells accordingly. IL-10, which indicating the immunosuppression, also dramatically increased in the whole process. Collectively, we hypothesis that there are two strategies involved in the JEV mediated immune evasion. On the one side, upregulation of the co-inhibitory molecules on T cells and APCs induces cell apoptosis of the CD3+ T cells, CD4+ T cells and TFH cells; on the other side, IL-10 may regulate TFH cells and Tregs differentiation. As a consequence, JEV evades from immune clearance and gains entry into the CNS leading to neuropathogenisis.
Maintenance of multiple Nef functions limits and dictates pathways of immune escape (VIR1P.1147)
Authors: Maness Nicholas , Das Arpita , Weiler Andrea , Friedrich Thomas
Published: January 2023

CD8+ T lymphocytes (CD8TL) can effectively control SIV and HIV replication. CD8TL targeting of particular regions in the Nef globular core is associated with control of both viruses suggesting common mechanisms of control. Deep sequencing of SIV from nine Mamu-B*017:01+ macaques revealed distinct selection patterns in two Nef epitopes. Functional assays revealed that acute escape in one epitope impacted a conserved structural face of the Nef core and reduced MHC-I downregulation, rendering infected cells more susceptible to CD8TL against other epitopes. In contrast, acute mutations in a nearby epitope impacted Nef’s ability to downregulate Tetherin and CD28, both of which are modulated via Nef interactions with host clathrin adaptor proteins, with mutations impacting MHC-I anchor residues being most destructive. In fact, anchor residues in both epitopes are almost entirely conserved throughout primate lentiviral history, spanning more than 40 millennia suggesting selection for maintenance of function. These data suggest mechanisms by which Nef targeting CD8TL can contribute to viral control; by targeting conserved epitopes involved in fuctions that are critical for high-level viral replication in vivo. Our approach to understanding T cell mediated control of SIV revealed novel insights into Nef structure:function relationships and suggest that particular regions of the Nef protein might be attractive components of vaccines designed to induce potent CD8TL responses.
Dengue virus infection elicits highly polarized CX3CR1+ cytotoxic CD4+ T cells associated with protective immunity (VIR1P.1126)
Authors: Weiskopf Daniela , Bangs Derek , Sidney John , Kolla Ravi , de Silva Aruna , de Silva Aravinda , Crotty Shane , Peters Bjorn , Sette Alessandro
Published: January 2023

The frequency of infection with any of the four dengue viruses (DENV 1-4) has increased dramatically in the last few decades, and the lack of a vaccine has led to significant morbidity and mortality worldwide. Dengue virus is a pathogen with unusual pathogenesis and protective immunity is poorly understood. While DENV-specific CD8+ T cell responses have been extensively studied, the breadth and specificity of CD4+ T cell responses remains to be defined. In effort to define HLA-restricted CD4+ T cell responses resulting from natural infection with DENV, we endeavored to map T cell responses in individuals from Sri Lanka where DENV is hyper-epidemic. Characterization of the phenotype of the responding CD4+ T cells revealed a DENV specific T cell subset that is specifically expanded in donors carrying an allele associated with protection from severe DENV disease. Analysis of DENV-specific CD4+ T cells revealed that the virus specific cells were highly polarized, with a strong bias towards a CX3CR1+ Eomes+ Tbet+ perforin+ granzyme B+ CD45RA+ CD4 CTL phenotype. Importantly, frequency of these cells correlated with a protective HLA DR allele and we demonstrate these cells have direct DENV-specific cytolytic activity. We speculate that cytotoxic dengue-specific CD4+ T cells may play a role in control of dengue infection in vivo, and this immune correlate may be a key target for dengue virus vaccine development.