bluuebunny/crossref_metadata_2025 huggingface dataset:
Understanding the opposing roles of the adaptive immune system in stroke (THER7P.955)
Authors: Ortega Sterling , kong xiangmei , Meeuwissen Anouk , Noorbhai Ibrahim , Anderson Ashley , Chen Ding , Monson Nancy , Stowe Ann
Published: January 2023

Stroke is caused by a loss of blood flow to the brain, which results in permanent neurological injury. Murine models of ischemic stroke implicate the immune system as both contributor and suppressor of neuro-inflammation following brain ischemia. Using a 60-min transient middle cerebral artery occlusion model and a recall response assay, we have detected autoreactive immune responses to myelin, neuronal and glial peptides, suggesting a possible immune involvement in further brain inflammation. We previously found that repetitive hypoxic preconditioning (RHP) prior to stroke endogenously decreases infarct volumes and creates a unique, anti-inflammatory B-cell phenotype that may contribute to RHP-mediated reversal of neuropathology. Now, we show that B(RHP)cell adoptive transfer significantly reduces infarct volumes and CNS-antigen autoreactivity in CD4+ T-cells compared to PBS- or control B-cell treatments. B(RHP)cells exhibited increased production of IL10, a neuroprotective cytokine, and IFN-γ, a cytokine that potentiates the conversion of effector to regulatory T-cells that suppress neuropathological immune cells. Co-culture of B(RHP)cells with T-cells significantly converted naïve CD4 T-cells to Foxp3+CD25+ regulatory CD4 T-cells. Based on these observations, we hypothesize that RHP induces a novel subset of neuroprotective B-cells as an important regulatory arm of the adaptive immune system that can be potentially harnessed for immunotherapeutic strategies.
Cbl-b-deficient mice demonstrate alterations in T cell trafficking but retain sensitivity to the Multiple Sclerosis therapeutic agent FTY720 (THER7P.957)
Authors: Fujiwara Mai , Anstadt Emily , Khanna Kamal , Clark Robert
Published: January 2023

The variable response to treatment options in multiple sclerosis (MS) suggests the need for genetic-based personalized therapeutic approaches. CBLB gene polymorphisms have been identified in MS, and altered T cell function and aberrant responses to interferon-β in MS patients with a CBLB mutation have been recently reported. Cbl-b is an E3 ubiquitin ligase that regulates T cell activation and Cbl-b-deficient (Cbl-b-/-) mice show many T cell abnormalities also described in MS patients. This suggests that Cbl-b-/- mice may provide a novel approach for analyzing treatment options in patients with MS-associated CBLB mutations. We now report that Cbl-b-/- CD4+ T cells show significant trafficking-related abnormalities: decreased lymph node accumulation after adoptive transfer into RAG-1-/- mice; increased sphingosine-1-phosphate receptor 1 (S1P1) expression; and decreased CD69 expression compared to wild-type cells. These data imply that CBLB mutations may compromise the therapeutic efficacy of FTY720, an MS-approved drug that modulates S1P1 expression. Despite altered Cbl-b-/- T cell trafficking, we observed robust inhibition of EAE by FTY720 in Cbl-b-/- mice. These results not only document a novel role for Cbl-b in T cell trafficking but also indicate that FTY270 may be effective in EAE/MS through mechanisms other than T cell lymph node trapping. Moreover, our findings suggest that FTY720 treatment is still an appropriate choice in patients with MS-associated CBLB mutations.
Impaired expansion of CD11b+Gr-1+ cells from MyD88-deficient donor bone marrow cells aggravates graft-versus-host disease (TRAN2P.966)
Authors: Lee Young-Kwan , Ju Ji Min , Shin Dong-Mi , Min Chang-Ki , Choi Eun Young
Published: January 2023

Myeloid differentiation 88 (MyD88), an adaptor molecule in TLR-associated signaling, has recently been known to play a role in expansion/activation of CD11b+Gr-1+ myeloid-derived suppressor cells. To understand role of MyD88 expressed by donor-originating myeloid cells in GVHD development, we investigated dynamics of T cell-depleted bone marrow (TCD-BM) cells and gene-expressions of CD11b+Gr-1+ cells originated from Myd88-/- donor mice after transplantation into allogeneic BALB.B hosts with WT T cells. The allogeneic recipients of Myd88-/- TCD-BM and WT T cells showed aggravated GVHD, with significant loss of CD11b+Gr-1+ population, compared with BALB.B recipients of WT TCD-BM and T cells. Impaired expansion of Myd88-/- TCD-BM cells in BALB.B GVHD hosts was confirmed via in vivo tracking analysis. However, their expansion was intact in syngeneic C57BL/6 recipients, indicating that impaired CD11b+Gr-1+ cell expansion from Myd88-/- TCD-BM cells was specific to GVHD circumstance. In transcriptome analysis, CD11b+Gr-1+ cells of MyD88-/- TCD-BM origin exhibited higher expression of genes related to apoptosis and antigen-presentation than did those of WT TCD-BM origin. These results suggest that impaired expansion of the CD11b+Gr-1+ cells from Myd88-/- TCD-BM cells in allogeneic hosts is related to enhanced apoptosis in combination with enhanced differentiation into DC-like cells in the presence of activated allo-reactive T cells, and provide insight into the control of GVHD.
CCR5+ regulatory T cells induced by metastatic breast carcinomas migrate toward a gradient of CCL8 and accumulate in metastatic lung tissue (TUM9P.1016)
Authors: Halvorsen Elizabeth , Hamilton Melisa , LePard Nancy , Bosiljcic Momir , Young Ada , Wadsworth Brennan , Bennewith Kevin
Published: January 2023

Under homeostatic conditions, regulatory T cells (Tregs) mediate peripheral tolerance and prevent autoimmunity. Despite their crucial role in the regulation of immunity toward innocuous antigens, Tregs may also contribute to the growth and metastasis of breast cancer by suppressing anti-tumor immune responses. We are interested in developing therapies to inhibit intratumoral Treg infiltration to decrease the growth of primary and metastatic tumors. Using mammary carcinomas syngenic to BALB/c mice, we identified elevated levels of CCR5+ Tregs in the primary tumour and metastatic lungs relative to naive control tissues. Interestingly, we observed that C-C chemokine receptor type 5 (CCR5) is highly and selectively expressed by Tregs in metastatic lungs relative to other immune cell populations. CCR5+ Tregs largely co-express CTLA-4 and CD103, indicating a suppressive phenotype. The production of CCL8, an endogenous ligand of CCR5, was increased in the tumour and lungs, and CCL8-mediated migration of Tregs ex vivo was inhibited by the CCR5 antagonist Maraviroc. These data suggest that targeting CCR5 or CCL8 may be a viable therapeutic strategy to inhibit Treg accumulation during tumour progression to decrease metastatic tumour growth. Currently, there is a paucity of research involving the role of CCR5+ Tregs in tumour progression. We anticipate that this work will advance the generation of targeted, immune-based therapeutics for the treatment of metastatic breast cancer.
Development of vaccination approaches that reverse leukemia-induced tolerance and reduce leukemic burden (TUM9P.1020)
Authors: Manlove Luke , Vrieze Katherine , Manlove Kezia , Pauken Kristen , Schenkel Jason , Williams Richard , Jenkins Marc , Farrar Michael
Published: January 2023

Current therapies induce only transient responses in patients with BCR-ABL+ B Acute Lymphoblastic Leukemia. Thus augmenting immune responses to the BCR-ABL fusion peptide could be therapeutically beneficial. The fusion between BCR and ABL encodes a neo-antigen (called BAp) that stimulates an immune response, but this response does not eliminate leukemia. To characterize the immune response to BAp we examined BCR-ABL+ leukemic cells in mice. Principal Component Analysis demonstrated that a CD4+ T cell response driven by high CD40 and low PDL1 on leukemic cells inversely correlated with leukemic burden. Using BAp:I-Ab-specific tetramers we tracked BCR-ABL-specific CD4+ T cells in response to leukemia. Naive mice had a small population of BAp:I-Ab-specific T cells. Transfer of live leukemia resulted in modest expansion (55-fold) of BAp:I-Ab-specific T cells, which were largely diverted into FOXP3+ Tregs. Depletion of Tregs led to increased expansion of BAp:I-Ab specific T cells and protected mice from leukemia. Likewise, acute viral vaccination in the presence of BAp (but not infection with Listeria monocytogenes expressing BAp) induced expansion of Ly6C+ BAp:I-Ab-specific T cells, prevented Treg induction, and significantly reduced leukemic burden. Thus, BCR-ABL-specific T cells exist in non-leukemic mice but are tolerized in the presence of leukemia; this tolerance can be broken by Treg ablation or specific vaccination approaches, leading to protection against leukemia.
Emigration of tumor specific regulatory T cells from the bone marrow is triggered by S1P1 and correlates with their accumulation in breast tumors (TUM9P.1001)
Authors: Rathinasamy Anchana , Dettling Steffen , Ge Yingzi , Umansky Ludmila , Herold-mende Christel , Domschke Christoph , Schütz Florian , Beckhove Philipp
Published: January 2023

High regulatory T cell (Treg) infiltration in breast tumors is associated with reduced survival. However, the source of tumor infiltrating Treg and signals underlying their migration from lymphoid organs to the tumor tissue remain elusive. We here demonstrate that pronounced Treg infiltration in human breast tumors correlates with a selective reduction of tumor antigen specifc Treg from the bone marrow. Using MHC-II tumor peptide tetramers we furthermore show that tumor specific bone marrow Treg selectively express Sphingosine-1-phosphate receptor 1 (S1P1), the receptor that mediates cell egress. S1P1 was upregulated in Treg upon TCR stimulation mediated by bone marrow resident antigen presenting cells and triggered selective Treg but not conventional T cell migration in response to S1P gradients between bone marrow and blood which we found to be significantly increased in breast cancer patients. Taken together, our data suggests a crucial role for bone marrow antigen presenting cells in regulating the surface expression of S1P1 on tumor specific Treg in the bone marrow which may represent an important source of tumor infiltrating Treg.
Nuclear TRAF3 Inhibits CREB-mediated survival and metabolic reprogramming in B lymphocytes (TUM10P.1045)
Authors: Mambetsariev Nurbek , Lin Wai , Stunz Laura , Hanson Brett , Arkee Tina , Bishop Gail
Published: January 2023

TRAF3 is an adaptor protein that negatively regulates signaling through CD40 and BAFF receptor in B lymphocytes. B cells isolated from B cell conditional TRAF3-/- mice display a remarkable pro-survival phenotype compared to wild type B cells, but the mechanism for this abnormal survival is poorly understood. We find that CREB protein expression and activity - but not mRNA - were increased in TRAF3-/- B cells. Inhibition of activity of CREB and its co-activator molecule CBP attenuated the survival of TRAF3-/- B cells. Immune precipitation revealed that TRAF3 associated with both CREB and CBP preferentially in the nucleus in B cells. The TRAF-C domain was identified as necessary and sufficient for TRAF3 nuclear localization. The human TRAF3 mutant LP1 isolated from a myeloma tumor and lacking a TRAF-C domain, failed to localize to the nucleus or associate with CREB. Guided by results of microarray analysis, we found that the proteins Glut1 and Hexokinase II, two CREB targets important for glucose metabolism, were elevated in TRAF3-/- B cells and associated with enhanced glucose uptake and broad metabolic reprogramming in Seahorse extracellular flux analysis. We are currently investigating the importance of TRAF3-regulated metabolic changes to B cell survival.
Investigation of Mf-mediated suppression of B cell activation (TUM10P.1048)
Authors: Riggs James , Valiuskyte Kornelija
Published: January 2023

We have previously shown that C57BL/6J (B/6) peritoneal cavity (PerC) macrophages (Mfs), via IFNg-triggered iNOS expression, reduce T cell activation promoted by mitogens (ConA) or by TCR ligation (anti-CD3e, SEB, etc). We wanted to assess whether PerC B cells were also susceptible to this form of myeloid suppression. B/6 PerC B cell responses triggered by BCR {F(ab’)2 anti-IgM} and TLR4 (LPS/TLR4L) were both suppressed and could be liberated by blocking prostaglandin production with indomethacin; neutralization of IL10 also increased the LPS response. Suppression of the BALB/c (B/c) PerC B cell response to BCR ligation was similar but the TLR4 response was not suppressed. Where the addition of exogenous IL4 or IL13 suppressed the B/6 response to BCR ligation, IL4 increased this response in B/c mice. Most interestingly, the PerC B cell response to CD40 ligation was not suppressed in either strain and addition of IL10 increased this response; addition of IL4 or IL13 suppressed the B/6 response but had no impact upon B/c B cells. Concurrent CD40 and TCR ligation revealed that iNOS-mediated suppression overrides the B cell response. Considering that the high myeloid:lymphoid cell ratio found in PerC cell cultures mimics that seen in tumor microenvironments, these results serve to inform strategies designed to promote anti-cancer immunity.
IL-22+CD4+ T cells promote colorectal cancer stemness via STAT3 transcription factor activation and induction of the methyltransferase DOT1L (TUM10P.1022)
Authors: Kryczek Ilona , Lin Yanwei , Nagarsheth Nisha , Peng Dongjun , Zhao Lili , Zhao Ende , Dou Yali , Owens Scott , Zgodzinski Witold , Majewski Marek , Wallner Grzegorz , Fang Jingyuan , Huang Emina , Zou Weiping
Published: January 2023

Little is known about how the immune system impacts human colorectal cancer invasiveness and stemness. Here we detected interleukin-22 (IL-22) in patient colorectal cancer tissues that was produced predominantly by CD4+ T cells. In a mouse model, migration of these cells into the colon cancer microenvironment required the chemokine receptor CCR6 and its ligand CCL20. IL-22 acted on cancer cells to promote activation of the transcription factor STAT3 and expression of the histone 3 lysine 79 (H3K79) methytransferase DOT1L. The DOT1L complex induced the core stem cell genes NANOG, SOX2, and Pou5F1, resulting in increased cancer stemness and tumorigenic potential. Furthermore, high DOT1L expression and H3K79me2 in colorectal cancer tissues was a predictor of poor patient survival. Thus, IL-22+ cells promote colon cancer stemness via regulation of stemness genes that negatively affects patient outcome. Efforts to target this network might be a strategy in treating colorectal cancer patients.
Recurrence of head and neck squamous cell cancer following resection may be predicted by assessment of immune infiltrates within the tumor (TUM10P.1056)
Authors: Paustian Christopher , Feng Zipei , Hulett Tyler , Moudgil Tarsem , Dubay Christopher , Campbell Mary , Welle John , Cheng Allen , Patel Ashish , Bui Tuan , Urba Walter , Leidner Rom , Bifulco Carlo , Bell Bryan , Fox Bernard
Published: January 2023

Tumor immune infiltrates are prognostic for many histologies, reinforcing the importance of the immune system in patient outcomes. Here we assessed a cohort of resected HPV+ and HPV- HNSCC tumors by multispectral imaging and a 770 gene digital transcript counting platform (NanoString nCounter system) that combines markers for 24 immune populations, 30 cancer antigens and relevant immune response genes. We performed IHC on FFPE and flowcytometry on enzyme-digested tumor allowing us to identify leukocyte subsets. Preliminary results show that while B cells, T cells and myeloid cells alternately predominate in different HNSCC tissue digests, NK cells rarely seen. Roughly half of our cohort has recurred at this time. Gene expression profiles indicate that tumors derived from patients with no evidence of disease contain more immune response related transcripts than tumors from patients that recur. Analysis of our discovery cohort by UPGMA hierarchical clustering indicates that the immune infiltrates in HNSCC may prove prognostic. Importantly, there are transcripts that may indicate a negative prognosis. Among these are a number of chemokines, TGFB2, the TLR4-TRIFF bridge TICAM2 and a molecule associated with anti-viral responses, IFITM1. Since these molecules constitute possible targets for therapeutic intervention, we are working to confirm their preponderance in tumors that recur by assessing a validation cohort as well as protein confirmation by IHC.
Distinct immunological activities of four variants of one mutation-generated neoepitope of a mouse fibrosarcoma (TUM10P.1023)
Authors: Brennick Cory , Duan Fei , Srivastava Pramod
Published: January 2023

Passenger mutations in cancer cells can give rise to mutant MHC Class I restricted epitopes, also referred to as neoepitopes. One such neoepitope, Tnpo3 (SYMLQALCI), has been described for the BALB/c Meth A fibrosarcoma, by our laboratory1. Immunization with a peptide corresponding to the Tnpo3 neoepitope elicits antigen specific CD8+ T cells, which are capable of mediating specific rejection of Meth A. Interrogating the site of mutation within the Tnpo3 neoepitope with NetMHC predicts three other variants of Tnpo3 having the same amino terminus but different carboxyl termini. Of these four Tnpo3 variants, three elicit functional CD8+ T cells upon immunization, and of these, only one elicits tumor rejection. Here, the structural and cellular immunological aspects of the differential activities of these four neoepitopes are investigated. 1Duan F, Duitama J, Al Seesi S, Ayres CM, Corcelli SA, Pawashe AP, Blanchard T, McMahon D, Sidney J, Sette A, Baker BM, Mandoiu II, Srivastava PK. Genomic and Bioinformatic Profiling of Mutational Neoepitopes Reveals New Rules to Predict Anticancer Immunogenicity." J Exp Med. 211 (2014): 2231-248. PMID: 25245761
Post-exposure treatment with neutralizing monoclonal antibodies prevents SHIV infection in infant rhesus macaques and limits establishment of latent viral reservoirs (VAC11P.1102)
Authors: Hessell Ann , Sacha Jonah , Kahl Christoph , Graham Barney , Mascola John , Haigwood Nancy
Published: January 2023

We have developed a unique pre-clinical SHIV/macaque model with the potential to address translational implications of early intervention in mother-to-child-transmission (MTCT). Based on prior passive studies in this model, we hypothesized that very early therapeutic treatment with potent NmAbs would limit or ablate establishment of viral reservoirs in babies born to HIV-infected mothers. Fourteen one-month-old rhesus macaques were inoculated orally with SHIVSF162P3. Cocktails of NmAbs PGT121 and VRC07-523 were prepared for subcutaneous passive transfer. A control group of two animals did not receive NmAbs. Beginning at 24 hours (1 day) post SHIV exposure, NmAbs were delivered subcutaneously and repeated at days 4, 7, and 10. Blood samples monitored for plasma viral load (PVL) and transferred NmAb kinetics. Cell-associated viral loads (CAVL) were measured in PBMC and in tissues at necropsy. Animals were followed for 24 weeks to assess PVL, PBMC-based CAVL, and T and B cell responses. Controls were infected by three d.p.i. NmAb-treated infants had undetectable PVL with no evidence of CAVL in PBMC or tissue samples at necropsy. ICS was used to detect Gag and Vif T cell responses. No anti-SHIV T cell responses were detected in lymphocytes isolated from inguinal lymph nodes at week 20 or in necropsy tissue samples. Results indicate early therapeutic treatment with a cocktail of potent NmAbs can prevent infection and limit the establishment of latent viral reservoirs.
Isolation of Dengue neutralizing antibodies from single cell sorted human antigen specific memory B cell cultures (VAC11P.1107)
Authors: Cox Kara , Tang Aimin , Chen Zhifeng , Horton Melanie , Meschino Steven , Yan Hao , Wang Xin-Min , Dubey Sheri , DiStefano Daniel , Vora Kalpit
Published: January 2023

Monitoring antigen-specific memory B cells and the antibodies they encode is important for understanding the specificity, breadth and duration of immune response to an infection or vaccination. The antibodies isolated could further help design vaccine antigens for raising relevant protective immune responses. However, developing assays to measure and isolate antigen specific memory B cells is technically challenging due to the low frequencies of these cells that exist in the circulating blood. Here we describe a method to develop a direct flow cytometric assay for the detection of Dengue envelope-specific memory B cells using a Dengue envelope specific mouse hybridoma line. We used this flow cytometry assay and a cultured B ELISPOT assay to enumerate Dengue-envelope specific B memory cells from a cohort of Dengue seropositive donors. Furthermore, we were able to culture the single sorted Dengue-envelope specific memory B-cells and differentiate them into plasma cells using cell lines expressing CD40L. The culture supernatants were assayed for antigen binding and the ability of the antibodies to neutralize the cognate dengue virus. Moreover, we successfully isolated the heavy and light Ig sequences and expressed them recombinantly as full length antibodies to reproduce the activity seen in culture supernatants. In conclusion, we have established a robust methodology to enumerate antigen-specific memory B-cells and assay their encoded antibodies for functional characterization.
Antibody-mediated toxin aggregation correlates with the ability of heterodimeric camelid antibodies to passively protect mice against ricin (VAC11P.1108)
Authors: Herrera Cristina , Tremblay Jacqueline , Shoemaker Charles , Mantis Nicholas
Published: January 2023

Ricin belongs to the A-B toxin family that also includes Shiga and cholera toxins. While immunity to ricin is antibody (Ab)-mediated, it is unclear what functional qualities of Abs are associated with immunity to ricin, considering that Abs with potent toxin-neutralizing activity (TNA) in vitro are not always protective in vivo. We recently produced a collection of alpaca heavy-chain only Abs (VHHs) specific for ricin’s enzymatic (RTA) and binding (RTB) subunits. To better define the attributes associated with immunity to ricin, we engineered VHH homodimers and heterodimers and characterized them in vitro and in vivo. Dimeric VHHs had 10-fold improved binding affinities for ricin and had 2-3 times increased TNA as compared to their monomeric constituents. However, neither improved affinity nor increased TNA correlated with a VHH’s ability to protect in vivo. Instead, immunity was associated with heterodimers composed of an RTA and an RTB monomer in which at least one had TNA. Analytical ultracentrifugation showed that all five protective heterodimers identified in this study promoted ricin aggregation in solution. The same five VHHs also prevented ricin from binding to cell surfaces in vitro. We propose that toxin aggregation and/or inhibition of attachment are likely key determinants of antibody-mediated protective immunity to ricin and possibly other A-B family members.
Preliminary assessment of hepatitis B-specific cellular immunity amongst a longitudinal vaccine cohort demonstrates evidence of natural exposure. (VAC11P.1110)
Authors: Simons Brenna , Spradling Philip , Zanis Carolyn , Choromanski Tammy , Case Samantha , Bruce Michael , Bruden Dana , Murphy Trudy , Knall Cindy , McMahon Brian
Published: January 2023

Duration of protection after hepatitis B (HB) vaccination is unknown; therefore, we examined humoral and cellular immunity among persons living in an area endemic for HBV, 30 years post-vaccination. Measurement of antibody to hepatitis B surface antigen (anti-HBs) indicated 51% (n=217) of persons retained anti-HBs titer ≥10mIU/mL. However, of persons who lost anti-HBs (<10mIU/mL) and received a booster dose, 89% responded. Our objective was to conduct a preliminary comparison of cellular and humoral immunity characteristics between persons who retained (n=28) anti-HBs levels ≥10mIU/mL and those who did not (n=18). Immune cell frequency was assessed by flow cytometry (B cell, T cell, memory, Treg, PD-1, CD57, NK/NKT). PD-1hiCD3+ lymphocytes were significantly increased (p = 0.047) in persons who maintained anti-HBs titer ≥10mIU/mL. Peripheral Blood Mononuclear Cells (PBMC) were stimulated directly ex vivo with whole HB Core Antigen or HB Surface Antigen and assessed for cytokine response. Results indicated no significant difference in the magnitude of HB Surface Antigen-specific responses in persons who maintained anti-HBs ≥10mIU/mL and those who did not. HB Core Antigen-specific TNFα responses were significantly higher (p=0.01) in persons who had lost anti-HBs titer. The observed cellular responses to HB Core suggest natural exposure amongst the cohort. Understanding cellular immunity afforded by the HB vaccine is critical to determining long-term protection.
A broadly protective anti-influenza neuraminidase monoclonal antibody (VAC11P.1100)
Authors: Hashem Anwar , Doyle Tracey , Li Changgui , Van Domselaar Gary , Wang Junzhi , Hurt Aeron , Brown Earl , Li Xuguang
Published: January 2023

Hemagglutinin (HA) and neuraminidase (NA) are the major surface glycoproteins of influenza viruses and the main targets of vaccine-induced antibodies (Abs). While several broadly neutralizing anti-HA Abs can cross-protect against diverse influenza subtypes, NA-specific Abs could only protect partially against strains from the same subtype. Through comprehensive bioinformatics analyses of all publicly available influenza A and B NA sequences, we found a universally conserved 9-mer peptide (ILRTQESEC) amongst all influenza NA proteins (amino acids 222-230). Growth kinetics of recombinant viruses with single alanine substitutions within this epitope proved its crucial roles in viral fitness and replication. Importantly, a monoclonal Ab (HCA-2 mAb) raised against this sequence showed broad in vitro inhibition against multiple strains from all influenza A NA subtypes (N1-N9) and influenza B lineages. It also provided in vivo heterosubtypic protection against lethal doses of H1N1 and H3N2 strains. Amino acid residues I222 and E227, located in close proximity to the active site, were found to be indispensable for inhibition by HCA-2 mAb. These findings reveal the essential role of this highly-conserved sequence in NA function and viral replication and show that it is sufficiently exposed to allow access of inhibitory Abs during the course of infection. Thus, it could represent a potential target for novel antivirals or vaccines against diverse strains of influenza A and B viruses.
Immune checkpoint blockade activates effective anti-tumor immunity in an orthotopic model of glioblastoma (VAC12P.1119)
Authors: Klein Sarah , Gokhale Praful , Jones Kristen , Saur Amy , Rodig Scott , Liao Xiaoyun , Kohl Nancy , Reardon David , Freeman Gordon
Published: January 2023

Glioblastoma presents a formidable obstacle for developing immunotherapeutic strategies due to a highly immunosuppressive systemic and micro-environment. Antibody blockade of co-inhibitory receptors CTLA-4 and PD-1 has been shown to overcome immunosuppression in clinical trials of various solid tumors. We evaluated the anti-tumor efficacy of murine antibodies targeting CTLA-4 and/or PD-1 in the GL261 orthotopic, immunocompetent mouse model of glioblastoma. We observed tumor regression and long-term survival in 15% and 50% of mice treated with anti-CTLA-4 or anti-PD-1 as single agents, respectively. Combination therapy resulted in 75% of animals surviving long-term and tumor-free. Importantly, similar efficacy was observed with both early (day 7) or late (day 14) onset of treatment, thus this therapeutic strategy was effective on established tumors. Furthermore, resistance to tumor re-challenge demonstrated the generation of immunological memory in treated mice. Upon investigating the systemic and tumor immunological profile, we discovered that combination therapy increased CD8+ and NK effector cells and decreased suppressive immune cells at the tumor site and draining lymph node. In addition, combination therapy increased the serum levels of various monocyte chemo-attractant chemokines involved in immune activation. Our data provide strong support for the evaluation of immune checkpoint blockade in glioblastoma patients, and predicts potential prognostic serum biomarkers.
Anti-CD25 antibody and denileukin diftitox deplete regulatory T cells in ID8 ovarian cancer equally, but αCD25 promotes more T cell cytotoxicity that correlates with better survival (VAC12P.1112)
Authors: Drerup Justin , Liu Aijie , Hurez Vincent , Curiel Tyler
Published: January 2023

In ovarian cancer (OC) regulatory T cells (Tregs) block anticancer immunity, suggesting Treg depletion could be beneficial. In our phase 2 denileukin diftitox (DDT) trial we found no clinical efficacy in 19 OC patients despite Treg depletion. αCD25 depletes Tregs but reportedly only works as cancer prophylaxis. We found that αCD25 did not treat subcutaneous B16 mouse melanoma whereas DDT did, and both reduced Tregs equally. We further show that in intraperitoneal ID8 challenge, αCD25 depletes Tregs in ascites and strikingly extends survival better than DDT, the first demonstration that αCD25 is effective cancer treatment (vs prophylaxis). Treg suppression of T cell proliferation in vitro from ID8-bearing mice was equal after DDT or αCD25. DDT and αCD25 could thus target or modulate Treg subsets or other immune cells differentially, as neither is Treg-specific. Despite reduced clinical efficacy, DDT increased beneficial IFN-γ+CD4+ and ID8-specific CD8+ T cells and NK cells in ascites better than αCD25, but also increased pro-tumorigenic IL-17+ and TNF-α+ CD4+ T cells, whereas αCD25 did not. αCD25 increased numbers of ascites GzB+CD107a+CD8+ T cells whereas DDT did not, suggesting increased cytotoxicity that could explain better clinical efficacy. These observations show that Treg depletion agents available for clinical use have distinct effects on antitumor immunity, help define clinical indications for αCD25 versus DDT and identify responsive cancers or anatomic compartments.
Strong systemic cancer immunostimulatory therapies cause stress-induced acute thymic involution (VAC12P.1114)
Authors: Sckisel Gail , Mirsoian Annie , Tietze Julia , Monjazeb Arta , Blazar Bruce , Wiltrout Robert , Murphy William
Published: January 2023

Agonistic immunotherapeutic regimens are being increasing applied in cancer. The thymus, and therefore naïve T cell output, is highly sensitive to stress environments. We therefore investigated thymic function following stimulatory cancer regimens (CpG, high-dose IL-2, anti-CD40) and found that acute thymic involution (TI) occurred in young mice within days of treatment initiation and recovered by two weeks post treatment cessation. Acute TI was hallmarked by a spike in systemic corticosterone levels and adrenalectomy prevented the involution suggesting it was mediated by glucocorticoids. Cancer patients are typically aged and, with obesity being a major risk factor, becoming increasingly obese as well. Both aging and obesity are associated with premature immune senescence, notably chronic TI. Data investigating the recovery from acute TI in the context of ongoing chronic TI are lacking since the vast majority of preclinical models involve the use of young mice (2-4ms). Treatment of obese and aged mice also resulted in acute TI. Using a congenic bone marrow adoptive transfer model, we tracked thymic recovery across young lean, obese, and aged models to show that recovery differed in magnitude and quality of T cells in the obese and aged. These data have profound implications for the long term risks of delayed thymic recovery and naïve T cell output which can increase susceptibility to infections in aged and obese cancer patients following systemic immunostimulatory regimens.
Differential Fc-receptor engagement drives an anti-tumor vaccinal effect (VAC13P.1123)
Authors: DiLillo David , Ravetch Jeffrey
Published: January 2023

Passively-administered anti-tumor mAbs mediate killing of tumor targets via FcγR-mediated cytotoxicity, a rapid and short-term process. However, anti-tumor mAb treatment can also induce a vaccinal effect, in which mAb-mediated tumor death induces a long-term anti-tumor cellular immune response. To determine how such responses are generated, we utilized a murine model of a mAb-mediated anti-tumor vaccinal effect. Using mice with a conditional deletion of FcγRs on dendritic cells (DCs), we demonstrate that FcγR expression by antigen-presenting DCs is required to generate an anti-tumor T cell response upon ADCC-mediated tumor clearance by anti-tumor mAb. However, human FcγR expression differs from mice; humans express two activating FcRs, FcRIIA and FcRIIIA, on cytotoxicity-mediating NK cells and monocytes, while antigen-presenting DCs only express FcRIIA. Thus, we used FcγR-humanized mice, which express the full array of human FcγRs in place of mouse FcγRs, to demonstrate that anti-tumor huIgG1 must engage huFcγRIIIA on monocytes/NK cells to mediate ADCC, but also engage hFcRIIA on DCs to generate a potent vaccinal effect and anti-tumor cellular immune response. Thus, while next-generation anti-tumor antibodies with enhanced binding to only hFcγRIIIA for ADCC are now in clinical use, optimal anti-tumor antibodies must be optimized for both their short-acting cytotoxic effects as well as for engagement of hFcγRIIA on DCs to stimulate long-term anti-tumor cellular immunity.
Human cytomegalovirus reprograms human bone marrow progenitor cells into nitric oxide-producing, long-life, immune suppressive monocyte subset to achieve latency (VIR9P.1142)
Authors: Zen Ke , Pan Chaoyun , Zhu Dihan , Liu Fenyong , Zhang Chen-Yu
Published: January 2023

It is generally regarded that human cytomegalovirus (HCMV) achieves latency in human haematopoietic progenitor cells. However, the nature of host cell at HCMV latent infection and the mechanisms governing the differentiation of progenitor cells mediated by HCMV remain elusive. In the present study, we identify that nitric oxide (NO)-producing, long-life, and immune suppressive monocyte subset differentiated from human bone marrow CD34+ progenitor cells serve as the true host for HCMV latency. This monocyte subset can suppress T cell proliferation and activity via NO-based mechanism. Both gain-of-function and lose-of-function assays show that high level NO production in progenitor cells via inducible nitric oxide synthase during HCMV infection is a key to suppress HCMV immediate early (IE) protein expression and viral replication to induce HCMV latency. NO production, correlating with progenitor cell differentiation, is promoted by signal transducer and activator of transcription 3 (STAT3) which activity is increased by HCMV infection. As a control, HCMV strain with US28 deletion, which cannot activate STAT3, fails to produce NO and achieve latency in haematopoietic progenitor cells. Our results demonstrate for the first time that HCMV achieves latent infection through reprograming human haematopoietic progenitor cells into NO-producing, long-life, immune suppressive monocyte subset.
Temporal regulation of T cell priming during peripheral infection (VIR10P.1167)
Authors: Mueller Scott , Hor Jyh Liang , Heath William
Published: January 2023

The in vivo dynamics of CD4 and CD8 T cell priming in draining lymph nodes (dLN) following infection and the types of interactions that occur with different dendritic cell (DC) subsets remains unclear. Immune responses to pathogens that remain localized to peripheral tissues and do not directly infect the lymphoid organs critically rely upon migratory DC to traffic antigen to dLN. Following Herpes Simplex virus infection (HSV-1) migratory CD11b+ DC transport viral antigens from the skin to the dLN and then transfer antigen to LN-resident CD8a+ DC for CD8 T cell priming. Although migratory DC can present antigens to CD4 T cells ex vivo, which DC subset(s) activate CD4 T cells in vivo, when and where CD4 T cells provide ‘help’ for CD8 T cell priming and the dynamics of these interactions are not known. Using a HSV-1 flank infection model combined with intravital 2-photon imaging of the draining inguinal LN to concurrently visualize pathogen-specific CD4 and CD8 T cells we find temporally separated priming of CD4 versus CD8 T cells. Early priming of CD4 T cells involved clustering with migratory DC and more rapid responses by these T cells, followed by later interactions of CD8 T cells with LN-resident DC that resulting in priming. Together, these data suggest unexpected temporal differences in T cell activation by DC subsets following localized viral infection.
Mapping the interaction footprint of Viral Immunoevasins on a “platform” MHC-I molecule using solution NMR spectroscopy (APP2P.102)
Authors: Sgourakis Nikolaos
Published: January 2023

Mouse cytomegalovirus encodes immunoevasin proteins that interfere with MHC-I antigen presentation to CD8+ T cells and natural killer (NK) cells. Members of the m02-m16 family contribute to viral survival and infectivity. One member of this family (m04) binds MHC-I molecules in the endoplasmic reticulum and accompanies them to the cell surface, while the related m06 binds and directs MHC-I to endosomes. Despite many genetic and functional studies, the structural basis of the specificities and functions of these immunoevasins is poorly understood due largely to difficulty in co-crystallization with their MHC-I ligands. To bypass this bottleneck we have previously determined the structure of the m04 core domain, using a hybrid methodology that combines sparse datasets recorded from Nuclear Magnetic Resonance (NMR) spectroscopy with sophisticated Rosetta macromolecular modeling. The structure revealed a divergent Ig-like fold that is unique to this family of imunoevasins, with long loops that provide flexible scaffolds for MHC binding. Here, we employ solution NMR to map he interaction footprint of m06 on MHCI-I. We make use of a previously engineered α1α2 molecule based on H2-Ld that shows improved NMR spectral features and binding properties. This study provides insight into a key site on MHC-I “discovered” by the virus and helps us to understand the function of this important viral family with respect to T and NK cell MHC recognition.
Preliminary analysis of the contributions of major histocompatibility complex-H2 to autoantibody production in a murine model of systemic lupus erythematosus. (BA3P.116)
Authors: Ruck Melissa , Hermiston Michelle
Published: January 2023

The stochastic and heterogeneous nature of Systemic Lupus Erythematosus (SLE) supports a model whereby multiple genetic and/or environmental hits culminate in loss of tolerance and autoantibody production. Consistent with this model, the phenotype of CD45E613R mice that contain a single point mutation in the juxtamembrane wedge of CD45 depends on genetic context. Despite similar dysregulated phosphatase activity in all immune cells, CD45E613R mice on a C57Bl/6 (B6) background have no overt phenotype while BALB/c (BA) mice develop anti-double stranded (ds) DNA antibodies. An unbiased genome-wide screen for modifiers of autoantibody production between CD45E613R B6 and BA mice identified two candidate loci: Wedge Associated Modifier (Wam) 1 on Chromosome (Chr) 9 encompassing tlr9 and Wam2 on Chr 17 encompassing MHC H2. Previous work has shown that the hyporesponsive BA TLR9 allele permits anti-ds DNA antibodies while the B6 TLR9 allele is resistant. Here, we analyze the contribution of the MHC to this phenotype. CD45E613R BALB/c mice congenic for the B6 H2 locus develop anti-Sm antibodies while B6 mice congenic for the BALB/c H2 locus develop anti-Sm and anti-dsDNA antibodies. H2 congenic CD45E613R BALB/c mice have increased transitional B cells and a trend in increased B1 cells compared to congenic B6 mice. Thus, we hypothesize that MHC influences autoantibody specificity.
Type 1 interferon signaling alters tolerance by promoting self-reactive B cell survival in the germinal centers (BA3P.106)
Authors: Domeier Phillip , Soni Chetna , Elias Melinda , Shwetank .. , Schell Stephanie , Rahman Ziaur
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by high titers of somatically mutated autoantibodies (AutoAbs) that are produced by self-reactive B cells aberrantly selected within the germinal centers (GCs). Type 1 Interferon (IFNa) signaling is implicated in AutoAb mediated pathogenicity in SLE. However, a defined mechanism by which IFNa signaling may regulate the GC checkpoint is unknown. To understand how IFNa signaling regulates the spontaneously developed GC (Spt-GC) response and associated AutoAb production, we utilized B6.Sle1b mice that harbor the lupus-prone NZM2410 strain-derived SLAM locus. These mice exhibit elevated Spt-GC and follicular helper T cell (Tfh) responses that associate with impaired B cell selection in GCs. B6.Sle1b mice deficient in IFNα receptor1 (B6.Sle1b. IFNaR1-/-) had significantly fewer Spt-GC B cells and Tfh cells than B6.Sle1b mice, and exhibited defects in B cell proliferation in vitro. These defects correlated with decreased viability of the IFNaR1-/- B cells, indicating that GC B cell survival requires IFNaR1 signaling. Histological analysis suggests that follicular dendritic cells may produce IFNa in the GC to promote B cell survival. The reduced GC numbers in B6.Sle1b.IFNαR1-/- mice resulted in markedly decreased total IgG Ab titers, including self-reactive AutoAbs. Together, our findings indicate that IFNa signaling helps aberrantly selected GC B cells survive in the Spt-GCs leading to AutoAb production.
Dysregulation of type II interferon associated mediators precedes autoantibody accumulation and type I interferon elevation and predicts transition to systemic lupus erythematosus classification (BA3P.109)
Authors: Munroe Melissa , Fife Dustin , Zhao Yan , Lu Rufei , Robertson Julie , Guthridge Joel , Niewold Timothy , Tsokos George , Keith Michael , Harley John , James Judith
Published: January 2023

Autoantibodies and interferon (IFN) are associated with clinical illness in systemic lupus erythematosus (SLE), but how these factors influence autoimmune disease development is unknown. This study evaluates the timing and changes in SLE-linked autoantibody specificities, serum IFN-α activity, and IFN-associated soluble mediator levels in the years preceding SLE classification. Serial sera from 55 SLE cases spanning pre- and post-SLE classification (average time = 4.3 years) and matched healthy controls (HC) were obtained from the Department of Defense Serum Repository. Cases with positive serum IFN-α activity had more SLE-linked autoantibody specificities (p<0.05). Growth curve modeling and path analysis corroborated that most autoantibody specificities precede IFN-α activity, and IFN-γ and IP-10 were elevated prior to or concurrent with autoantibody positivity (p<0.01). The number of autoantibody specificities and IFN-associated soluble mediators, as well as female gender, predicted increases in IFN-α activity (p<0.04; ANCOVA). IFN-γ and MCP-3 levels, as well as the presence of anti-chromatin and spliceosomal autoantibodies, best predicted transition to SLE with 93% accuracy >4 years before SLE classification (Random Forest). These data indicate that pre-clinical immune system perturbations allow for the accumulation of autoantibodies and subsequent elevation in IFN-α activity immediately preceding clinical symptoms and SLE classification.
Fatal autoimmunity results from the conditional deletion of Snai2 and Snai3 (BA4P.121)
Authors: Pioli Peter , Chen Xinjian , Weis Janis , Weis John
Published: January 2023

Transcriptional regulation of gene expression is a key component of orchestrating proper immune cell development and function. One strategy for maintaining these transcriptional programs has been the evolution of transcription factor families with members possessing overlapping functions. Using the germ line deletion of Snai2 combined with the hematopoietic specific deletion of Snai3, we report that these factors function redundantly to preserve the development of B and T cells. Such animals display severe lymphopenia, alopecia and dermatitis as well as profound autoimmunity manifested by the production of high levels of autoantibodies as early as three weeks of age followed by death within 30 days of birth. Autoantibodies included both IgM and IgG isotypes and were reactive against cytoplasmic and membranous components. A regulatory T cell defect contributed to the autoimmune response in that adoptive transfer of wild type regulatory T cells alleviated symptoms of autoimmunity. The results demonstrated that appropriate expression of Snai2 and Snai3 in cells of hematopoietic derivation plays an important role in development and maintenance of immune tolerance.
Loss of peripheral protection in pancreatic islets by proteolysis-driven impairment of VTCN1 (B7-H4) presentation is associated with the development of autoimmune diabetes (BA4P.132)
Authors: Radichev Ilian , Maneva-Radicheva Lilia , Amatya Christina , Salehi Maryam , Ellefson Jacob , Savinov Alexei
Published: January 2023

Antigen‑specific activation of T cells is an essential process in the control of effector immune responses. Defects in T cell activation, particularly in the co‑stimulation step, have been associated with many autoimmune conditions including type 1 diabetes (T1D). Recently, we demonstrated that V-set domain-containing T cell activation inhibitor-1 (VTCN1) protein is reduced on antigen-presenting cells from diabetes-susceptible NOD mice and human T1D patients. Here, we show existence of a similar defect in the pancreatic islets, as both α and β cells gradually lose their VTCN1 protein during T1D development despite the up‑regulation of the VTCN1 gene. Diminishment of functional islet cells’ VTCN1 is caused by the metalloproteinase NRD1 and leads to a significant induction of proliferation and cytokine production by diabetogenic T cells. Inhibition of NRD1 activity, on the other hand, stabilizes VTCN1 and dulls the anti-islet T cell responses. Therefore, we suggest a general endogenous mechanism of defective VTCN1 function, which affects both lymphoid and peripheral target tissues during T1D progression and results in aggressive anti-islet T cell responses. This mechanism is tied to up-regulation of NRD1 expression and likely acts in two synergistic proteolytic modes: cell-intrinsic intracellular and cell-extrinsic systemic. Our results highlight an importance of VTCN1 stabilization on cell surfaces for the restoration of altered balance of immune control during T1D.
Increase of endosomal Sec61 enhances antigen cross-presentation and induces immune glomerular injury: a novel ‘self-organized criticality theory’ explaining the cause of systemic lupus erythematosus (BA4P.129)
Authors: Tsumiyama Ken , Shiozawa Shunichi
Published: January 2023

Objective: We found that systemic lupus erythematosus (SLE) was induced by repeatedly immunizing the mice with antigen, and have proposed a novel ‘self-organized criticality theory’ explaining the cause of SLE. CTL generated via antigen cross-presentation induces lupus tissue injuries. Here we examine the molecular mechanism of antigen cross-presentation in relation to lupus nephritis. Methods: Bone marrow-derived DC (BMDC) and splenic DC (spDC) were cultured with OVA and an inhibitor of Sec61 Exotoxin A (Exo A) to examine the localization of early endosome antigen 1 (EEA1), calnexin, Sec61 and OVA. Sec61 was immunoprecipitated from DC, followed by detection of OVA. OVA in cytoplasm was also detected. Mice were repeatedly immunized with OVA and Exo A. Sec61 in endosome of spDC was detected using immunoprecipitation of EEA1. Results: In DCs, OVA was co-localized with an endosomal marker EEA1, and subsequently separated from EEA1. OVA was not co-localized with ER marker calnexin. Instead, OVA was co-localized and co-precipitated with Sec61. OVA was detected in cytoplasm of BMDC, however, Exo A abolished the detection of OVA. In spDC of mice developed glomerular injury, not only Sec61 in endosome but also OVA in cytoplasm were increased. Further, co-immunization with OVA and Exo A abrogated the development of glomerular injury. Conclusion: Increase of Sec61 in endosome promotes antigen cross-presentation and is essential for the development of glomerular injury likely of SLE.