bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel class of host-derived etiological agent for autoimmunity: oligomers of endogenous proteins bind to self-nucleic acids and trigger type I IFN production by plasmacytoid dendritic cells (44.2)
Authors: Di Domizio Jeremy , Zhang Ran , Zhuo Ming , Stagg Loren , Ladbury John , Gilliet Michel , Cao Wei
Published: January 2023

Under certain conditions, some soluble endogenous proteins can form ordered oligomeric structures leading to the assembly of stable insoluble amyloids, a process that increases with age. The presence of such structures is associated with cellular toxicity and diseases development, e.g. Alzheimer disease, type II diabetes. Recent studies clearly show that the primary toxic species in such pathologies is the soluble oligomers of proteins, precursors of amyloids. Yet, it is unknown if such aberrant forms of proteins would elicit any immune reaction. Here we obtained oligomers derived from several human endogenous proteins and characterized their biochemical and immunological functions. The oligomeric proteins preferentially bind to both DNA and RNA and can be effectively internalized by cells. Surprisingly, the soluble protein oligomers enable prominent IFNα production by PBMCs to self-DNA, self-RNA and necrotic cell debris in a pDC-dependent manner. Consistently, peptides from amyloid β and prion protein, two known etiological agents associated with amyloid diseases, display similar innate immune functions by complexing with nucleic acids and inducing IFN production by pDCs. Therefore, oligomers of endogenous proteins formed during the aging process may strongly promote the host’s type I IFN response to self-nucleic acids, a reaction likely favoring the development of autoimmunity, such as SLE where strong type I IFN presence correlates with disease pathology.
Granulocyte expansion and mobilization following EAE induction is correlated with G-CSF production. (44.43)
Authors: Rumble Julie , Rao Praveen , Segal Benjamin
Published: January 2023

Experimental autoimmune encephalomyelitis (EAE) is a murine model of neuroinflammatory demyelination that is frequently used as a model for multiple sclerosis (MS). EAE is induced mice by immunization with a myelin epitope and adjuvants. We have previously shown that granulocytes are critical for blood-brain-barrier (BBB) breakdown and clinical disease in EAE. In the current study we investigated the pathways by which granulocytes are regulated during EAE. We found that granulocytes expand in the bone marrow and the bloodstream by day 3 p.i., which was preceded by a spike in systemic levels of G-CSF, a granulocyte growth and mobilization factor. Administration of Bordetella pertussis toxin (BPT) is required for the clinical manifestation of disease in our model. Interestingly, sustained expression of G-CSF and accumulation of circulating granulocytes only occurred in mice given BPT. Treatment of immunized mice with an antagonistic G-CSF receptor-Fc fusion protein prevented clinical EAE. We postulate that induction of G-CSF drives the expansion and mobilization of bone marrow-derived granulocytes that ultimately mediate BBB breakdown and subsequent CNS infiltration by leukocytes. Consistent with our findings, individuals with MS experienced severe exacerbations upon the administration of recombinant G-CSF following bone marrow transplantation. Furthermore, we suggest granulocyte growth and mobilization factors as novel therapeutic targets in autoimmune demyelinating disease.
Limiting HCDR3 diversity abrogates the antibody response to the bacterial polysaccharide α 1→3 Dextran (45.20)
Authors: Mahmoud Tamer , Schroeder Harry , Kearney John
Published: January 2023

Anti-polysaccharide antibody responses in mice are often oligoclonal and the mechanisms involved in the production and subsequent antigen-driven selection of these clones are poorly understood. In this study, we investigated the α 1→3 Dextran (DEX) antibody response of gene-targeted mice limited to the usage of one DH gene segment (D-limited mice). We show that the DEX-specific antibody response is broadly comparable in D-limited mice to that of wild type BALB/c (WT) mice. D-limited mice forced to use DH reading frame 2 (ΔD-DµFS) showed both a higher total anti-DEX antibody response compared to other D-limited mice and the dominant DEX-specific J558 idiotype (Id) was higher compared to all mouse groups. Nucleotide sequencing of HCDR3s of DEX-specific plasmablasts showed that ΔD-DµFS mice generated 70% of clones similar to those from WT, including the predominant adult molecularly-determined clone J558. All three D-limited mouse groups showed a higher frequency of unusual DH-less HCDR3 regions compared to WT suggesting extensive junctional modification mediated by TdT. D-limited mice on TdT-deficient background failed to elicit a response to DEX. Overall, these data show that TdT activity is required to compensate for constraints on HCDR3 diversity for the generation of a normal anti-DEX antibody response by D-limited mice.
Defining the molecular and cellular niche for CD8 memory T cell maintenance by 4-1BB and 4-1BBL (46.18)
Authors: Lin Gloria Hoi Ying , Snell Laura , Wortzman Michael , Vidric Mariana , Surh Charles , Watts Tania
Published: January 2023

Following the resolution of an infection, memory lymphocytes can persist for the lifetime of the animal and play a critical role in protection from re-infection. Using mixed BM chimeras, we show that 4-1BB on the T cells is required for maximal CD8 memory T cell response to influenza virus. 4-1BB is constitutively expressed on a fraction of CD8 memory T cells in the BM of unmanipulated mice, with much lower expression in the spleen and LN. The common gamma chain cytokines IL-2, IL-15, and IL-7 can each induce 4-1BB expression on CD8 CD44Hi cells in the absence of Ag ex vivo. However, using CD122-/- mixed BM chimeras and IL-7Ra antibody blockade, they were found to be dispensable for in vivo expression of 4-1BB on memory CD8 T cells. In contrast, the TNFR family member GITR plays a non-redundant role in inducing 4-1BB expression on CD8 memory T cells. 4-1BBL can be detected on CD11c+ cells in the spleen and BM and on CD45 negative cells in the BM. We found that 4-1BBL on radioresistant cells provides the major source of 4-1BBL to maintain the CD8 memory T cell pool detected in the BM, whereas the absence of 4-1BBL on radiosensitive cells makes an additional contribution to the CD8 memory pool detected in the spleen. This study defines a specific niche in the BM, where 4-1BB is expressed on CD8 memory T cells in a GITR-dependent manner and 4-1BBL on a radioresistant CD45 negative cell in the BM provides the source of ligand for 4-1BB-dependent memory CD8 T cell maintenance.
Gliomas display complex cell surface topographies that resist cytolytic lymphocytes but are reversed by using fascin siRNA (48.2)
Authors: Jadus Martin , Hoa Neil , Ge Lisheng , Kuznetsov Yurii , McPherson Alex , Cornforth Andrew , Ahmed Nabil , Lamb Lawrence
Published: January 2023

Gliomas are invasive cancers that resist all forms of attempted therapy, but immunotherapy has improved survival for some patients. We present evidence that another level of complexity may also contribute to lack of responses by the lymphocytes towards gliomas. Atomic force microscopy of four different glioma types: human U251 and rat T9 and F98 glioma cells, including freshly isolated human GBM neurosphere cultures (containing “stem cell-like cells’), revealed a complex surface topography with numerous microvilli and filopodia. These structures were not found on other cell types. Electron microscopy and immunofluorescence microscopy of glioma cells confirmed that microvilli are present. U251 cells with microvilli resisted the cytolytic actions of different human effector cells, (lymphokine-activated killer cells, γδ T cells, conventional cytolytic T lymphocytes, [CTL], and chimeric antigen receptor redirected T cells) better than their non-microvilli expressing counterparts. Killer cells released perforin which was detected within the glioma’s microvilli/ filopodia, indicating these structures can receive the cytolytic effector molecules, but cytotoxicity is suboptimal. Transfection of fascin siRNA into U251 cells prevented the microvilli from forming and allowed cytolytic cells to kill these adherent cells just as well as the non-attached glioma cells. These microvilli play multiple roles in glioma biology, such as invasion and resistance to lymphocyte-mediated killing.
Dissociation of porcine reproductive and respiratory syndrome virus neutralization from antibody production to major envelope protein peptides (49.22)
Authors: Li Juan , Murtaugh Michael
Published: January 2023

Porcine reproductive and respiratory syndrome (PRRS) is the most severe infectious disease facing the swine industry worldwide. The etiologic agent is PRRSV, an enveloped arterivirus. GP5 and M, the major envelope proteins, form disulfide-bonded heterodimeric complexes that are involved in PRRSV entry into porcine alveolar macrophages (PAM). Previous studies identified several neutralization epitopes in GP5 polypeptide, containing amino acid residues (AA) 29-52, and potential unknown neutralization epitopes in M protein. To test whether PRRSV-neutralizing antibodies (NA) are directed to GP5 and M ectodomain polypeptides, we constructed three recombinant single-chain proteins: the two predicted ectodomains of GP5 (GP5-5’), the two ectodomains of M (M-5’), and all four ectodomains (GP5-M). In particular, GP5-5’ contains the reported neutralization epitopes. We found that the convalescent pig sera after PRRSV infection contained various levels of antibodies to those proteins, but the Ab titers did not correlate with the NA response. Immunization of pigs with these recombinant proteins did not induce detectable NA response. Finally, protein-specific Abs that were purified from PRRSV-neutralizing serum by affinity chromatography showed no neutralizing activity, whereas the protein-specific Ab-depleted sera retained similar neutralizing activity as the original sera. The results, therefore, do not support that PRRSV-NA are directed to the ectodomain polypeptides of GP5/M.
Dendritic cells control CD4+CD25+ Treg cell function in vitro through juxtacrine delivery of IL-2. (50.3)
Authors: Field Elizabeth , Kulhankova Katarina , Rouse Todd , Nasr Mohamed
Published: January 2023

CD4+CD25+ regulatory T cells (Tregs) require IL-2 for their expansion and function. Unable to produce IL-2, Tregs rely on IL-2 from a neighboring cell. We explored whether DCs might serve as an important source of IL-2 for Tregs. Though a minor subset of DCs make IL-2, the IL-2 producing DCs were preferentially found in conjugate formation with Tregs. DCs continued to transcribe IL-2 in the presence of Tregs, but protein secretion was completely diverted to the Treg. Uptake of DC-derived IL-2 by Tregs involved cell:cell contact and CD25. DC IL-2 increased Tregs levels of CD25 and foxp3. Surprisingly, Treg suppressor function in vitro depended on DC IL-2. Tregs in cultures with WT DCs suppressed proliferation of CD4+ Teff more effectively than Tregs in cultures with IL-2 KO DCs. Tregs also suppressed IL-2 secretion by CD4+ Teff if cultured with WT DCs but showed no suppression if cultured with IL-2 KO DCs. In the later cultures, the transient addition of IL-2 at levels ≥ 500 U/ml restored Treg suppressor function, suggesting that DC to Treg delivery of IL-2 was spatially controlled to achieve locally high concentrations of IL-2. The findings demonstrate a clear requirement for DC-derived IL-2 for optimal Treg suppressor function in vitro and reveal a novel model of juxtacrine delivery of IL-2 from DCs to Tregs. The data lead us to speculate that IL-2 producing DCs may play a role in initiating the activation of functional Tregs in vivo through spatial delivery of signal 3.
Phenotyping human Tregs: the key to understanding autoimmunity? (50.6)
Authors: Magid-Bernstein Jessica , Rohowsky-Kochan Christine
Published: January 2023

T regulatory cells (Tregs) are important in the development and progression of autoimmunity. Tregs are usually identified by their expression of CD25 and Foxp3, although functionally and phenotypically diverse subsets exist. By identifying various subsets we hope to clarify their function and role in autoimmunity. We are also studying the novel cytokine IL-35, which is believed to be integral for Treg function. To identify Treg subsets we analyzed expression of CD27, CD127, and CD39 on CD4 T cells with varying CD25 levels. A CD27+CD127- phenotype correlates highly with CD25 and Foxp3 expression. CD39 expression is higher on CD4+CD25hi T cells than on CD25med/lo cells. We cultured sorted Treg subsets with T effector (Teff) cells to measure suppression. High expression of CD25 was a better marker for suppressive ability than the CD27+CD127- phenotype. CD39+ Tregs did not suppress proliferation when activated with anti-CD3 and anti-CD28, but they did inhibit IFN-γ production. Expression of the IL-35 subunits, EBI3 and p35, was observed in activated Tregs but not resting or activated induced Tregs or Teffs. Studies are ongoing to examine IL-35 in Treg subsets. Elucidating Treg mechanisms and subsets will enable us to clarify how Tregs suppress, how they are defective in autoimmunity, and ultimately how this defect can be overcome to treat autoimmunity.
Immunogenicity of a Group A Streptococcal peptide vaccine conjugated to CRM197 (53.12)
Authors: Caro-Aguilar Ivette , Ottinger Elizabeth , Hepler Robert , Nahas Deborah , Wu Chengwei , Joyce Joseph , Heinrichs Jon , Skinner Julie
Published: January 2023

Group A Streptococcus (GAS) causes several disease manifestations and remains a huge medical burden in developing and developed countries, therefore there is an imminent need for a prophylactic vaccine. One focal point of vaccine development for GAS has been the M protein, an alpha-helical coiled-coil surface protein. An epitope derived from the C-terminal region of the M protein, J8, has been shown to be highly immunogenic and to generate opsonophagocytic killing (OPK) activity and protection in mice. Here we evaluated the use of an alternative protein carrier, CRM197, a molecularly detoxified version of diphtheria toxoid (DT), by conjugating J8 to CRM197 and immunizing C3H and Balb/c mice with this vaccine on AAHSA (Amorphous Aluminum Hydroxyphosphate Sulfate Adjuvant). J8-specific ELISA and whole bacteria immunofluorescence (IFA) were used to evaluate immunogenicity. Functional antibody activity was evaluated by opsonophagocytosis (OPK) and protection from a GAS intranasal challenge. J8 conjugated to CRM197 induced high antibody responses to J8; these specific antibodies recognized the epitope on the surface of the bacteria by IFA and demonstrated functional activity in an OPK assay. Additionally, J8-CRM197 immunized mice demonstrated significantly greater survival than adjuvant control immunized mice. This study showed that CRM197 is a suitable carrier protein for J8 peptide conjugation in C3H and Balb/c mice.
Soluble IL-7 receptor (sCD127) in pediatric HIV infection (57.9)
Authors: Faucher Sylvie , Decker Wendy , Sherring Alice , Bogdanovic Dragica , Bergeron Michele , Mandy Francis , Lapointe Normand , Soudeyns Hugo , Sandstrom Paul
Published: January 2023

IL-7 is an essential cytokine in T-cell development and homeostasis. It binds to the IL-7R receptor, a complex of the IL-7Rα (CD127) and common γ (CD132) chains. There is significant interest in evaluating the expression of CD127 on human T-cells as it often decreased in medical conditions leading to lymphopenia. A soluble CD127 (sCD127) is released in plasma and may contribute to disease pathogenesis through its control on IL-7 activities. We determined the concentrations of soluble CD127 in plasma of children infected with HIV using a quantitative bead-based sCD127 immunoassay. Eleven children were followed over a period of up to two years. The sIL-7R detected in HIV infected children ranged from 66 to 1004 ng/mL. Individual sCD127 concentrations remained stable when measured serially during a period of up to two years in five children with low viral load (HIV RNA 50-500 copies/mL). In children with uncontrolled viral load (n=6) (HIV RNA 50-137 000 copies/mL), the concentrations of sCD127 varied by up to 4 fold.This is the first report on the quantification of plasma sCD127 in pediatric HIV infection. The concentrations of sCD127 in children varied by up to one log. We previously reported a similar variation among adult sCD127 concentrations. The stability of the sCD127 concentrations in children with low viral load and the variations observed in children with uncontrolled viral load warrant further studies on the sCD127 as a potential marker of T cell homeostasis.
Participation of B-1 cells in the nasal influenza vaccine-induced antibody production. (59.10)
Authors: Kouro Taku , Iida Ryuji , Hattori Yuki , Shinoda Kaori
Published: January 2023

Aim: Administration of vaccine via nasal route has many merits including induction of cross-protective IgA on the surface of respiratory mucosa. However there is little information about IgA producing cells in the nasal mucosa. We examined role of B-1 cells in nasal vaccination because B-1 cells are responsible for production of natural antibodies which possess wide reactivity to various pathogens. Results: B-1 cell deficient BtkKO mice and control were anesthetized and intranasally administrated with influenza HA vaccine with poly(I:C) as an adjuvant twice in 2-week interval. 2 weeks later, BtkKO mice showed significantly reduced anti-HA nasal IgA response compared to control. In contrast, when splenocytes from vaccinated mice were re-stimulated with inactivated influenza virus, cells from BtkKO mice showed same or higher response than controls in the terms of Th1 cytokine production and IFN-γ secreting cell number. Further, reconstitution of B-1 cells in the BtkKO mice resulted in partial recovery of Ig response to vaccination. Discussion: Although Btk is expressed not only in B cells but also in myeloid cells including dendritic cells, defect of B-1 cells should be responsible for the reduced nasal Ig response in the BtkKO mice because T cells in these mice were properly activated. Thus B-1 cells and their unknown regulators will be good target of potent nasal vaccine adjuvant.
Tumor-induced regulatory B cells promote cancer metastasis by inducing generation of FoxP3+ regulatory T cells (66.16)
Authors: Olkhanud Purevdorj , Bodogai Monica , Wejksza Katarzyna , Biragyn Arya
Published: January 2023

The involvement of regulatory and suppressive B cell has been mostly associated with control of autoimmune responses; and their role in cancer escape remains unknown. Recently we reported the existence of a unique subset of B cells that suppress T cell responses to promote lung metastasis of breast cancer. These cells, designated as tumor-evoked B cells (tBreg), were actively generated by tumor cells from normal B cells. Interestingly, number of FoxP3+ Tregs was also enhanced in tumor-bearing mice, although the mechanism of Treg expansion remains unknown. We hypothesized that tBregs induce the generation of Tregs. Utilizing in vitro and in vivo approaches, we demonstrate that tBregs indeed induce the generation of FoxP3+ Tregs from resting CD4+ T cells. The molecular mechanism of this process is that tBregs abundantly express and utilize TGFb. The generation of FoxP3+ Tregs is blocked by selective inhibition of the TGFb receptor signaling, such as by the use of TGFb-type I receptor-like kinase ALK5 inhibitor. Importantly, the tBreg-induced FoxP3+ Tregs promote lung metastasis, as proved by their adoptive transfer in NOD/SCID mice. Taken together, our data indicate that tBregs facilitate lung metastasis by inducing Tregs in a TGFb-dependent manner. Thus, the clinical implications of our findings are that, to effectively combat cancer and its metastasis, tBregs have to be controlled to interrupt the initiation of cancer-induced suppressive events.
Phenotypical and functional changes in myeloid-derived suppressor cells during the tumor progression: FKBP51 regulates the suppressive function of MDSCs (66.25)
Authors: Kim Yun-Sun , Kim Yeon-Jeong , Kim Eun-Kyung , Lee Jung-Mi , Seo Jeong-Hwan , Park Young-Jun , Kang Ho-Woong , Kang Chang-Yuil
Published: January 2023

Immature myeloid cells which are increased by tumor-derived factors actively suppress immune cells. Therefore, they are called as myeloid-derived suppressor cells (MDSCs). Since the effect of cancer immunotherapy would be enhanced by removing MDSCs or inhibiting their suppressive function, we tried to find a target for MDSC regulation. MDSCs were increased during the tumor progression and MDSCs of late stage tumor bearing mice had stronger suppressive activity than those of early stage tumor bearing mice, in T cell proliferation assays. To find factors that change the MDSCs during the tumor progression, we analyzed gene expression profiles in MDSCs at various tumor stages and found that expression of Fkbp5 was increased in the Mo- and PMN- MDSCs of late stage tumor bearing mice. By means of rapamycin, which inhibit the PPIase function of FKBP51 protein, we demonstrate that the FKBP51 was involved in the suppressive function of MDSCs via regulation of iNOS expression. In addition, the FKBP51 enhanced the survival of MDSCs. By targeting the FKBP51, we may regulate the suppressive function of MDSCs and enhance the effect of anti-cancer immunotherapy.
Cross-regulation of innate response in human PBMC by an enveloped RNA virus and LPS (67.11)
Authors: Dhanushkodi Nisha , Mohankumar Vidyarani , Pokkali Supriya , Raju Ramaswamy
Published: January 2023

The innate response to polymicrobial infections and their pathways are poorly understood. In this work, we used Sindbis virus (SIN), an enveloped RNA virus and LPS as inducers of innate response. PBMC from healthy volunteers were exposed to SIN Toto 1101, in the presence and absence of E.coli LPS. We observed absence of new virus release, SIN/GFP reporter expression and viral RNA synthesis suggesting poor replication of SIN in human PBMC. However, significant lymphocyte activation, cytokine and chemokine release were seen upon exposure of PBMC to SIN. Analysis of lymphocyte activation markers showed that, LPS was a better inducer of CD69 expression in CD4, CD8, and CD19 lymphocytes than SIN. Increased secretion of IL-12p40, IL-1β, TNF-α, IL-6, IL-18, IL-10, IL-8, RANTES, MCP-1, MIP1-α and MIP1-β was induced by LPS whereas, SIN induced IL-6, IFN-α, MCP-1, MIP1-α, MIP1-β, I-TAC and IP-10 secretion. SIN downregulated LPS induced IL-12p40 secretion, whereas LPS inhibited virus induced IFN-α and IP-10. SIN also suppressed both the spontaneous and LPS induced secretion of IFN-α, IL-17, IL-13 and SLC from PBMC. To analyze involvement of specific PBMC subsets and associated signaling events, ELISPOT and phosphoflow are used. The absence and presence of antagonism between SIN and LPS for lymphocyte activation and secretion of select cytokines and chemokines may help in understanding pathogenesis of mixed infections, and find applications in vaccine development and virotherapy.
A DV protein based in domine II and III of E protein expressed in plants induce an effective immune response (67.7)
Authors: López Moisés , Machorro Jazmin , Davila Enrique , Lim Miguel Angel , Barron Leticia
Published: January 2023

Dengue is the most important arboviral disease. Despite the efforts to develop an effective vaccine there is none available. In the virus the envelope protein is the most expose and it is the most immunity target. The E protein has 3 domains, the domain III (EIII) is involved in neutralizing Abs induction. Domain II (EII) contains a fusion peptide that may induce Abs with neutralizing activity. Recently Anti-DEC, has been considered as a potential adjuvant when fused with different viral proteins due to its use in targeting antigen to DCs. The goal of this work was to fuse genetically different sequences from domains II and III to heavy chain variable region of the Ab Anti-DEC-205 spaced by a flag sequence named OLLAS in order to improve the humoral response. The fusion sequences DEC-OLLAS-EII-EIII was cloned in the vector pICH31070. To express these DNA, the plants N. benthamiana were infiltrated and the protein expression was analyzed by W.B. at different days post-infiltration. The high expression was observed at day 8, then, the proteins were purify by preparative PAGE and refolded with L-arg buffer. Following groups of 8 mice were inoculated 3 times with 30μg of recombinant protein. The immunity of this protein was analyzed by ELISA, immobilizing DEN viral particles and important titer of antibodies were observed in the group of mice immunized with DEC-OLLAS-EIII-EII in contrast with the control group. Moreover neutralizing Abs was observed in the mentioned group
Dominant TNF-α Mycobacterium Tuberculosis-specific CD4 T-cell responses discriminate between latent infection and active disease (99.10)
Authors: Harari Alexandre , Rozot Virginie , Bellutti Enders Felicitas , Perreau Matthieu , Mazza Stalder Jesica , Nicod Laurent P. , Cavassini Matthias , Calandra Thierry , Lazor lanchet Catherine , Jaton Katia , Faouzi Mohamed , Day Cheryl L. , Hanekom Willem A. , Bart Pierre-Alexandre , Pantaleo Giuseppe
Published: January 2023

Background: Diagnosis of Mtb infection requires several parameters. IFNg-release assays do not discriminate between active TB disease and latent Mtb infection. Methods: Mtb-specific T-cell responses were investigated in a test cohort of 283 subjects with known diagnosis of latent Mtb infection or active TB disease and subsequently in a validation cohort of 114 subjects with blinded clinical status. Mtb-specific T-cell responses were analyzed by polychromatic flow cytometry using ESAT-6 and CFP-10 peptide pools. Results: Mtb-specific IFNg ELISpot responses were not different between patients with active TB disease or latent Mtb infection. In contrast, the functional profile of Mtb-specific CD4 T-cell responses was significantly different between active TB disease and latent Mtb infection (ESAT-6 or CFP-10, all P<0.0001) in the test cohort. Overall, Mtb-specific CD4 T-cell responses from patients with latent Mtb infection were TNFa+IFNg+IL-2+, while single TNFa-producing Mtb-specific CD4 T-cell responses were dominant in patients with active TB disease. Using this parameter (i.e. % of single TNFa-producing cells) as a diagnostic tool in an independent cohort of 101 patients with blinded TB diagnosis, we obtained >90% concordance between the clinical and the cytokines profile in predicting active TB disease and latent Mtb infection Conclusions: Polychromatic flow cytometry is a novel and reliable laboratory tool for the timely diagnosis of acute TB disease.
Presence of long lived bone marrow effector cells mediate protection against a virulent bacterial pathogen (99.30)
Authors: Anderson Rebecca , Crane Deborah , Bosio Catharine
Published: January 2023

Francisella tularensis is a virulent bacterium that causes lethal disease in humans. The cellular response that protects against a virulent Francisella infection (SchuS4) is not clearly understood. Live vaccine strain (LVS) vaccination in animal models confers nearly 100% protection against SchuS4 challenge; unfortunately, the mechanism of protection is not well defined. Therefore, the goal of this project is to determine the population and location of anti-Francisella effector cells that develop 3 months after LVS vaccination. The ability of cells from the spleen, bone marrow, liver, draining lymph node and blood of LVS vaccinated mice were assessed for limiting in vitro replication of SchuS4 in macrophages. Surprisingly, bone marrow cells from LVS vaccinated mice significantly reduced SchuS4 replication; however, cells from blood, spleen, liver, and lymph nodes did not control infection. The bone marrow is a unique environment that maintains memory B and T cells, as well as facilitates NK cell development. Therefore, CD90.2+ cells were purified, which represent B, T and NK cells. CD90.2+ cells reduced the total number of SchuS4 infected macrophages by nearly 50%, compared to CD90.2- cells. Upon further purification, CD49b cells elicited control of SchuS4 infected macrophages. These results suggest the effector population may be an NK, NKT, killer DC or T cell that mediates in vitro control of SchuS4 infection of macrophages.
Pseudomonas aeruginosa reduces the immune response to respiratory viruses (99.33)
Authors: Bomberger Jennifer , Ely Kenneth , Bangia Naveen , Berwin Brent , Green William , Enelow Richard , Stanton Bruce
Published: January 2023

P. aeruginosa chronically colonizes the respiratory tract of patients with chronic obstructive pulmonary disease, bronchiectasis and Cystic Fibrosis. These patients experience debilitating and life-threatening viral exacerbations of unknown etiology. Cif, a bacterial toxin secreted by P. aeruginosa, promotes the degradation of several ABC transporters involved in innate immunity, including CFTR and Pgp. The current study tests the hypothesis that Cif enhances the degradation of the transporter associated with antigen processing (TAP), resulting in reduced adaptive T-cell responses to viral pathogens. Cif inhibited the deubiquitinating enzyme, ubiquitin specific protease 10 (USP10), resulting in a time-dependent increase in the polyubiquitination and proteasomal degradation of TAP1. Cif inhibited USP10 activity by stabilizing an inhibitory protein-protein interaction between USP10 and G3BP1. The Cif-mediated decrease in TAP1 expression resulted in decreased MHC class I viral antigen presentation, CD8+ cytotoxic T lymphocyte (CTL) recognition of influenza virus-infected cells and dramatically reduced MHC class I cell surface levels on CD11c+ cells in the BAL of mice treated with P. aeruginosa. We propose that P. aeruginosa, by secreting the Cif toxin, inhibits the ability of CTL to recognize and eliminate viral infections, severely compromising the immune defenses of the lung. This is the first identified bacterial toxin that inhibits the viral immune response of the host.
Interleukin-10 limits RSV-induced disease severity by modulating the virus-specific T cell response (105.4)
Authors: Weiss Kayla , Christiaansen Allison , Fulton Ross , Meyerholz David , Varga Steven
Published: January 2023

The host immune response contributes to the severity of pulmonary disease following acute respiratory syncytial virus (RSV) infection. IL-10 is a potent immunomodulatory cytokine produced by multiple CD4 T cell subsets following RSV infection. We hypothesized that IL-10 negatively regulates the RSV-specific immune response thereby limiting disease severity. We observed that peak viral replication and clearance is not altered by the absence of IL-10 or following IL-10R blockade. However, disease severity as measured by weight loss and airway resistance is significantly increased in the absence of IL-10 or following IL-10R blockade following acute RSV infection. Histological analysis indicates that following IL-10R blockade there is an increase in mucus production and inflammation in the lung airways. We also observed a significant increase in pro-inflammatory cytokines and chemokines in the absence of IL-10 following RSV infection. Moreover, the total number of the IFN-γ-producing CD8 and CD4 T cells responding to the infection are significantly increased following IL-10R blockade. Interestingly, IL-10R blockade during RSV infection also altered CD4 T cell subset distribution, resulting in a significant increase in IL-17A-producing CD4 T cells and a concomitant decrease in Foxp3+ regulatory T cells. These results demonstrate that IL-10 plays a critical role in modulating the adaptive immune response to RSV by limiting T-cell-mediated pulmonary inflammation and injury.
Encounter with antigen-specific CD4 T cells promotes down-regulation of MHC-II in Dendritic cells (106.18)
Authors: Furuta Kazuyuki , Ishido Satoshi , Roche Paul
Published: January 2023

Major histocompatibility complex class II molecules (MHC-II) on dendritic cells (DCs) present antigenic peptides to CD4 T cells. MHC-II and T cell receptor (TCR)-mediated DC-T cell interactions induce activation and proliferation of naïve CD4 T cells. The fate of MHC-II molecules after engagement with TCR has not been extensively studied. In this study we investigated the effects of engagement of cell surface MHC-II with TCR. By incubating DCs with T cells, we found that activated CD4 T cells induced degradation of cell surface MHC-II in an antigen-specific manner. T cell engagement-induced degradation of MHC-II was also mimicked by crosslinking of MHC-II by anti-MHC-II mAb. After MHC-II crosslinking, clustering and endocytosis of MHC-II were induced and MHC-II was transported to lysosomes and degraded. MHC-II clustering and endocytosis were inhibited by cell surface cholesterol-depletion using methyl β-cyclodextrin (MβCD). Although ubiquitination is known to regulate cell surface expression of MHC-II in DCs, by using ubiquitination site-deficient MHC-II mutant mice we found that ubiquitination was not required for crosslinking induced MHC-II degradation. Finally, we found that crosslinking of MHC-II on DCs inhibited their ability to stimulate additional naïve CD4 T cells. These results demonstrate that CD4 T cells promote the down-regulation of MHC-II and limit antigen presentation by inducing endocytosis and degradation of MHC-II in DCs.
Gauging the contribution of endogenous MHCII pathways to influenza-specific CD4+ T cell responses (106.32)
Authors: Miller Michael , Luckashenak Nancy , Eisenlohr Laurence
Published: January 2023

The current paradigm for MHC Class II peptide generation holds that exogenous antigen enters the endosomal/lysosomal pathway, where it is processed and presented on the cell surface for presentation to CD4+ T cells. Increasing evidence from our lab and others has elucidated pathways that process endogenous proteins for presentation on MHC Class II and operate exclusively during live infection. Despite this, the components of these pathways, the prevalence of such epitopes, and their general contribution to the immune response remain poorly characterized. Using an IFN-γ-ELISpot assay, we screened a peptide library of the flu proteome to identify potential endogenous CD4+ epitopes generated following live or UV-inactivated virus priming. The differences in specificities were striking, with “live only” hits compromising a major fraction of the response and mapping to both structural and non-structural proteins. Experiments are ongoing to identify those epitopes that can be strictly categorized as endogenous, and to identify cellular components (proteasome, TAP, H-2M,…) that contribute to their generation. A comprehensive understanding of endogenous MHCII epitopes and their corresponding presentation pathways could contribute substantially to improved vaccine design and efficacy.
Aminoglycoside-induced readthrough of stop codons in vivo elicits presentation of novel immunogenic MHC class I epitopes . (106.39)
Authors: Robinson Tara , Zook Matthew , Goodenough Elliot , Nelson Chad , Flanigan Kevin , Atkins John , Howard Michael , Eisenlohr Laurence
Published: January 2023

Aminoglycoside antibiotics are known to induce stop codon readthrough in eukaryotic cells, and thus hold potential for the treatment of genetic diseases caused by nonsense mutations. A potential consequence of aminoglycoside treatment is the translation of 3’ noncoding regions and generation of major histocompatibiltiy complex (MHC)-restricted neoepitopes, a potential cause of autoimmune reactivity. Here we explored the impact of aminoglycoside-induced readthough on the production of MHC class I binding peptides. Firstly, we developed a panel of recombinant vaccinia viruses in which expression of a MHC class I binding peptide depends upon stop codon readthrough. Both in vitro and in vivo studies indicated that gentamicin induces activating levels of epitope with the magnitude of the response strongly influenced by identity of the stop codon and the surrounding context in agreement with a previously determined hierarchy. Secondly, we treated uninfected cells with gentamicin to assess the level of aminoglycoside-induced readthrough on native stop codons. HPLC analysis revealed a small number of differences in the peak profiles. Further analysis will be done with mass spectrometry to determine the identity of these epitopes, and where they are located in the mouse genome. The data from these and future studies can be used determine the extent to which stop codon readthrough occurs, and the implications for eliciting autoimmunity.
Variable processing and presentation of HIV epitopes in HIV-infectable cell subsets (106.8)
Authors: Dinter Jens , Gourdain Pauline , Lai Nicole , Shimada Mariko , Duong Ellen , Le Gall Sylvie
Published: January 2023

HIV-specific CD8 T cells (CTL) play a major role in reducing viral load in acute HIV infection and in the spontaneous control of HIV viremia. HIV infects CD4 T cells, monocytes, macrophages and dendritic cells. Whether these subsets have equivalent capacity to produce and present MHC-I restricted epitopes is unknown. We analyzed the effect of TLR agonist-mediated maturation on the processing and presentation of antigens in monocyte-derived dendritic cells (DC) and macrophages (MP). LPS-induced maturation decreased significantly the proteasomal tryptic activity and lysosomal activities in DC whereas in mature MP these activities were increased. Using cytosolic extracts of DC and MP as a source of peptidases we showed by mass spectrometry that degradation of 3-ISW9 in LPS-matured DC yielded 2-fold more epitope HLA-B57-restricted ISW9 than in immature DC. Using recombinant p24 as an exogenous antigen cross-presented by DC or MP expressing HLA-B57 we observed reduced killing of cells by ISW9-specific CTL compared to TW10- or KF11-specific CTL. The lower ISW9-specific killing of target cells was partly rescued by preincubation of cells with protease inhibitor, suggesting that lower cross-presentation may be due to higher intracellular degradation of ISW9. Differences in antigen processing activities in DC and MP upon maturation, and differences in HIV sequences sensitivity to intracellular degradation may affect the production and presentation of HIV epitopes to CTL.
IRF8 Interacts with the BCL6 Co-repressor, BCOR (109.5)
Authors: Yoon Jeong Heon , Feng Jianxun , Morse III Herbert
Published: January 2023

BCOR has been discovered as a BCL-6 interacting co-repressor, but little is known about its biological activities in normal B cell development and function. Previously, we found that the interferon regulatory factor 8 (IRF8), also known as interferon consensus sequence-binding protein, ICSBP, directly targets a large number of genes in germinal center (GC) B cells including BCL6. In this study, we screened potential binding partners of IRF8 using a retrovirus-based protein complementation assay in a mouse pre-B cell line. We found that IRF8 interacts directly with BCOR and the alpha helical region of IRF8 is required for this interaction. Using a siRNA-mediated IRF8 knockdown mouse B cell lymphoma cell line, we showed that IRF8 represses BCOR and enhances BCL6 transcription. Taken together, these data suggest that a complex comprised of BCOR-IRF8 modulates BCL6 associated transcriptional regulation of B cell function. Additional studies to evaluate the possible role of BCOR-IRF8 complex are in progress.
Antigen availability is a critical determinant of in vivo CD8+ T cell-dendritic cell interaction kinetics and initiates effector/memory fate decisions (110.21)
Authors: Henrickson Sarah , Senman Balimkiz , Quigley Michael , Alexe Gabriela , Stutte Susanne , Jesneck Jonathan , Iannacone Matteo , Flynn Michael , Imam Sabrina , Mazo Irina , Haining William , von Andrian Ulrich
Published: January 2023

Naïve CD8+ T cells are activated through interactions with antigen (Ag) bearing dendritic cells (DCs) in lymph nodes (LNs). Multiphoton intravital microscopy (MP-IVM) of LNs has demonstrated that T cell activation occurs in three phases. The duration of the initial phase of transient, serial DC-T cell interactions is inversely related to Ag dose. It had been assumed that the second phase, characterized by stable DC-T cell contacts, is necessary for T cell activation, however, here we show that this is not always the case. T cells interacting with DCs presenting Ag at low abundance and with a short half-life did not transition from phase one to phase two, whereas a higher dose of the same Ag yielded robust phase two transition. T cells exposed to either antigenic constellation proliferated and developed equivalent effector functions, including cytokine production and cytotoxicity, over the first two days after priming. There are profound differences in the transcriptome at 12h and 24h after activation between CD8 T cells exposed to antigenic constellations that did or did not promote phase two interactions. Only T cells that had undergone stable contacts gave rise to memory, whereas activating conditions that did not support stable contacts yielded immunological amnesia. T cells can make fate decisions in vivo within a few hours after Ag exposure that result in long-term memory or abortive effector responses and correlate with the kinetics of T cell-DCs interactions.
Mechanisms that affect long-distance activity of the 3′ regulatory region of the immunoglobulin heavy chain gene locus (111.31)
Authors: Volpi Sabrina , Chatterjee Sanjukta , Ju Zhongliang , Verma-Gaur Jiyoti , Hassan Rabih , Emelyanov Alexander , Feeney Ann , Birshtein Barbara
Published: January 2023

The 3' regulatory region (3' RR) of the Igh locus works at long distances on VH and I region promoters to initiate germline transcription (GT) and promote class switch recombination (CSR). The 3' RR contains multiple elements, including enhancers (hs3a-hs4), and a proposed insulator region containing CTCF binding sites, i.e. hs5/6/7 and the downstream region (“38kb”). To better understand how the 3' RR functions, we used chromatin immunoprecipitation assays to monitor transcription factor binding sites in splenic B cells that undergo GT and CSR (LPS+/-IL4), or are deficient in GT and CSR (p50-/-), or do not undergo CSR despite efficient GT (anti-IgM+IL4). The hs5-7 and 38kb regions were observed to contain multiple Pax5 binding sites and Pax5 was shown to interact with CTCF. We found that the Pax5 binding profile to the 3' RR dynamically changed during CSR. The role of the hs5-7 region was assessed by genomic deletion. B cells from hs5-7 KO mice showed a modest increase in expression of the nearest downstream genes. In addition, Igh alleles in KO mice were in a less contracted configuration compared to WT Igh alleles and showed a two-fold increase in the usage of proximal VH7183 gene families. Hs5-7 KO mice were essentially indistinguishable from wild type mice in B cell development, allelic regulation, class switch recombination, and somatic hypermutation. These observations suggest redundancy and synergism among the multiple modules of the 3′ RR for Igh regulation.
Immunologic dysfunction underlies pathology in a mouse model of Rett syndrome. (111.52)
Authors: Cronk James , Derecki Noel , Djukic Aleksandra , Kipnis Jonathan
Published: January 2023

Rett syndrome is a devastating autism spectrum disorder, characterized by tremors, breathing dysregulation, impaired locomotion, and mental retardation. Most cases are caused by mutation of the X-linked gene, MECP2, which encodes a methyl-CpG-binding protein. Although MECP2 is expressed in many cell types, pathology has been generally attributed to neuronal dysfunction. However, we recently showed striking arrest of disease in the Mecp2-null mouse model of Rett by manipulation of the immune system. LysmCre-directed wild type MECP2 expression in myeloid cells of Mecp2-null mice and bone-marrow transplantation from wild type into Mecp2-null mice both markedly improved several disease sequelae. When peripheral myeloid cells from Mecp2-null mice were examined, they were significantly impaired in cytokine production (TNF and IL-10) and growth factors (IGF-1) after stimulation, and in phagocytosis. Moreover, peripheral organs from Mecp2-mutant mice showed significantly increased levels of monocytes and granulocytes, as well as a striking skew towards a naïve (CD62LHigh/CD44low) phenotype of T cells. In addition, spleens and livers of Mecp2-mutant mice are significantly smaller than those from wild type. Importantly, these findings correlated with data from human patients, who displayed similar hematologic changes. These data suggest that loss of Mecp2 may lead to immunologic dysfunction, which could underlie pathologies seen in Rett syndrome.
Calpain proteases are required for effective clearance of enterobacteria within the peritoneum of mice to prevent systemic infection (112.5)
Authors: Kumar Vijay , Hall Chris , Greer Peter , Craig Andrew
Published: January 2023

The ubiquitously expressed calpain 1 and 2 isoforms are heterodimers consisting of a catalytic subunit (capn1 or capn2), and a common regulatory capns1-encoded subunit, which is required for protease stability and activity. Although calpains regulate neutrophil and macrophage activation, their roles in immunity remain poorly defined. Here, we use fes-driven Cre recombinase (fes-Cre) to delete capns1 in mice, thus eliminating calpain 1/2 activity in Cre-expressing cells. We detect near complete capns1 deletion in hematopoietic cells, consistent with the tissue-specific expression pattern of fes. Next, we compared the response of control and capns1 targeted mice in the feces-in-peritoneum (FIP) model of sepsis (i.p. injection of enterobacteria). At 2-6 hours post infection, mice were sacrificed and bacterial load, neutrophil recruitment, and cytokine levels in peritoneal lavage fluid (PLF) and peripheral blood were compared between genotypes. We observed comparable levels of neutrophil infiltration and cytokine production in PLF from control and capns1 targeted FIP-treated mice. However, bacterial killing was highly reduced (>10-fold) in capns1 targeted compared to control mice; and this correlated with increased enterobacterial escape to the peripheral blood. These observations suggest that calpains are not required for inflammatory cytokine production or neutrophil recruitment, but they are required for bacterial clearance, and may protection against development of sepsis.
Modulation of DNA vaccine immunity by Fas-FasL signaling (113.15)
Authors: Yan Jiabin , Zapata Juan , Pauza C. David , Salvato Maria
Published: January 2023

The effects of apoptotic death on immunogenicity have been studied intensively, but conclusions about its benefits remain controversial. We hypothesized that inhibiting the Fas-FasL signaling pathway would enhance DNA vaccine potency. We were able to confirm and extend the findings from Norman Letvin’s group (Greenland et al, 2007) that Fas KO mice developed much better cellular immune response (CMI) to SIVmac gag in both quality and quantity. Delivered by electroporation, we co-injected B6 mice intramuscularly with plasmids expressing SIVmac gag and pro- or anti-apoptotic molecules. Our studies showed that the dose and ratio between antigen plasmid and apoptosis modifier were two key factors that influence CMI. More importantly, we found that these apoptosis-modifiers had different influences on the CMI of primary and secondary effectors. Both cFLIP and FADD had little influence on primary responses. However, after one boost, cFLIP enhanced CMI at 1:1 ratio while FADD enhanced CMI at 1:2 ratio, where they both down-regulated the Treg population. Unexpectedly, though shRNA-FasL enhanced primary responses, it inhibited secondary responses by up-regulating the Treg population. At a 1:4 ratio shRNA-Fas inhibited the primary response, but it had no influence on the secondary response. Furthermore, we found that Fas co-delivery impaired both primary and secondary CMI. Our results indicate that many circumstances contributing to Fas-FasL signaling affect DNA vaccine potency.