bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel antibody-like single-chain TCR antibody Fc fusion protein
Authors: Wong Hing C , Newman Robert G , Marcus Warren D , Liu Bai , Jeng Emily K , Alter Sarah , Rhode Peter R
Published: January 2023

We have previously reported the construction of a fusion protein composed of a soluble single-chain T cell receptor genetically linked to the constant domain of the human IgG1 heavy chain (TCR-Ig). The antigen recognition portion of the protein binds to an unmutated peptide derived from human p53 (amino acids 264–272) presented in the context of HLA-A2.1, whereas the IgG1 Fc provides effector functions. The protein is capable of forming dimers, specifically staining tumor cells, and promoting target and effector cell conjugation. The protein also has potent antitumor effects against p53+/HLA-A2.1+ human tumor xenografts in athymic nude mice and can mediate cell killing by antibody-dependent cellular cytotoxicity. Therefore, TCR-Ig behaves like an antibody, but possesses the ability to recognize antigens derived from intracellular targets. To test TCR-Ig in fully immunocompetent models, we have generated murine tumor cells stably expressing the human p53 epitope by constructing a single chain trimer composed of the human p53 peptide genetically linked to murine beta 2 microglobulin genetically linked to HLA-A2.1, with the inclusion of a disulfide trap to enhance stability. Utilizing haNK (NK-92 cells stably expressing high affinity Fc receptor and IL-2) as effector cells in the presence of TCR-Ig we demonstrated specific killing of murine single chain trimer expressing tumor cells. We are currently evaluating the antitumor activities and vaccinal effects of TCR-Ig in an HLA-A2.1 transgenic mouse model. TCR-Ig may represent a novel group of immunotherapeutics that has the potential to expand the range of tumors available for targeted therapies beyond those currently addressed by conventional antibody-based approaches.
Variegated gene expression and Sox-13 mediated regulation of WC1, a hybrid PRR/Co-receptor exclusive to memory gd T cells
Authors: Damani-Yokota Payal , Telfer Janice C , Baldwin Cynthia L
Published: January 2023

γδ T cells have broader reactivity and respond more rapidly compared to αβ T cells which are restricted to peptide bound to MHC. Some γδ T cells bear lineage-specific glycoproteins known as WC1 that are coded by a multigenic family composed of 13 members. Structurally, WC1 molecules have 6–11 SRCR domains, followed by a conserved transmembrane and a variable cytoplasmic tail. γδ T cells expressing these molecules can be further divided into subsets that express either WC1-3 or WC1-4, a dichotomy analogous to CD4 and CD8 molecules and similarly set up in the thymus. While WC1-3+ and WC1-4+ γδ T cells share a restricted repertoire of the γδ TCR genes, expression of WC1-3 vs. WC1-4 determines whether the γδ T cell is activated by Leptospira; reactivity correlates with expression of WC1-3 and the ability of its SRCR domains to bind to Leptospira. ShRNA-mediated knockdown of WC1-3 significantly reduces the ability of WC1-3 expressing γδ T cells to respond to the bacteria, guiding us to a hypothesis that WC1 is essential for recognition of bacterial pathogens by γδ T cells and that the WC1 family members expressed by a particular cell will determine its ability to respond to an infection. Evaluation of WC1 gene expression among individual γδ T cells indicates that WC1-3 and WC1-4 are always expressed reciprocally, establishing functional subsets. These γδ T cell subsets appear to be set in the thymus, alluding to the transcription factor occupancy of WC1-3 vs WC1-4. Occupancy of candidate Sox13 binding sites differ between WC1-3 and WC1-4 loci, suggesting that Sox13 may play an important role in determining the ability of mature γδ T cells to respond to bacteria.
Assessing the serological antibody repertoire to Flaviviruses in the endemic population of the Zika forest in Uganda
Authors: Demina Anna Vladimirovna , Lutwama Julius J , Hertz Tomer , Lobel Leslie
Published: January 2023

While the Zika virus has been suspected to cause complications in newborns of women infected during pregnancy, it has circulated in Africa for more --than half a century without any reported cases of microcephaly in children. To assess the prevalence of Zika infections in the Zika forest region we collected human sera samples from 81 people (17–56 years old), who live in Zika forest region, Entebbe, Uganda in 2016, and included both individuals hospitalized with acute febrile illness, as well as health individuals. Elisa assays were used to profile the IgM, IgG serological repertoire to Zika, Dengue 1-4 (DENV1-4) and Yellow Fever (YFV) viruses. We found that 17 individuals (21%) were positive for Zika, 7 of which were hospitalized with acute infection. The hospitalized patients had overall higher antibody titers to Zika. There also were serologically positive samples for: DENV1 n=11 (13.5%); DENV2 n=9 (11%), DENV3 n=4 (5%); DENV 4 n=4 (5%). 11 subjects (13.5%) were positive for YFV. Overall, 49% of the subjects had antibodies to at least one of these Flaviviruses, and 17% had antibodies to two or more Flaviviruses. While serological responses cannot accurately diagnose the specific Flavirvirus infections in these individuals due to significant cross reactivity within this viruses our data suggests that Flavivirus infections are very common in the Zika forest region. While a recent study reported that previous dengue infections may cause antibody dependant enhancement upon subsequent Zika infection, our data suggests that the Zika forest region in which many different Flaviviruses have been circulating for over 50 years may an important region to study such effects in the human population.
Development of a rapid diagnostic kit for antigen and antibody of Middle East respiratory syndrome coronavirus
Authors: Lee Eun-Young , Ko Hae Li , Jung Seo-Yeon , Nam Jae-Hwan
Published: January 2023

In 2015, there was an outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) in Korea. Therefore, rapid diagnostic kits for the viral antigen and antibodies against MERS-CoV are urgently required. First, we produced and purified a spike nanoparticle of MERS-CoV by the insect expression system. It was highly soluble and intact for immunization. The spike nanoparticle was used as a coating antigen for enzyme-linked immunosorbent assay (ELISA) to detect MERS-CoV-specific antibodies in the serum. The spike nanoparticle, at least with 1.5 ng/well as a coating antigen, can detect serially diluted standard antibodies purified from rabbits immunized with inactivated MERS-CoV, and from rats immunized with spike nanoparticle. Moreover, it can successfully detect MERS-CoV in patient serum. Next, to develop sandwich ELISA to detect MERS-CoV antigens, we generated 616 hybridoma clones and selected 37 monoclonal antibodies (MAbs), which exclusively reacted against the spike nanoparticle. Among them, three MAbs (S-15, S-16, and S-34) showed about 40% plaque reduction against MERS-CoV. Of the 37 MAbs, we selected the two that showed the optimal combinations to develop sandwich ELISA. This sandwich ELISA also can detect serially diluted MERS-CoV antigen. In this study, we developed two MERS-CoV-specific ELISA kits. One is for detecting MERS antibodies in patient serum and the other is for detecting MERS-CoV itself. Both ELISA kits can be reliably used for diagnosis of MERS-CoV.
Adaptive immune responses to Zika virus are important for controlling virus infection and preventing infection in brain and testes
Authors: Winkler Clayton W , Myers Lara M , Woods Tyson A , Messer Ron J , Carmody Aaron B , McNally Kristin L , Scott Dana P , Hasenkrug Kim J , Best Sonja M , Peterson Karin E
Published: January 2023

The recent association between Zika virus (ZIKV) and neurological complications including Guillain-Barré Syndrome (GBS) in adults and CNS abnormalities in fetuses highlights the urgency to understand the immunological mechanisms controlling this emerging infection. Studies have indicated that ZIKV evades the human type I IFN response suggesting a role for the adaptive immune response in resolving infection. However, the inability of ZIKV to antagonize the mouse IFN response renders the virus highly susceptible to circulating IFN in murine models. Thus, as we show here, although wild type C57BL/6 mice mount both cell-mediated and humoral adaptive immune responses to ZIKV, these responses were not required to prevent disease. However, when the type I IFN response of mice was suppressed, then the adaptive immune responses became critical. For example, when type I IFN signaling was blocked by antibodies in Rag1−/− immunodeficient mice, the mice showed dramatic weight loss and ZIKV infection in the brain and testes. This phenotype was not observed in Rag1−/− mice or mice treated with anti-IFNAR alone. Furthermore, we found that the CD8+ T cell responses of pregnant mice to ZIKV infection were diminished compared to non-pregnant mice. It is possible that diminished cell-mediated immunity during pregnancy could increase virus spread to the fetus. These results demonstrate an important role for the adaptive immune response in control of ZIKV infection, and imply that vaccination may prevent ZIKV-related disease, particularly when the type I IFN response is suppressed as it is in humans.
Dissecting the polyclonal antibody response of nurse sharks (Ginglymostoma cirratum) to immunization with influenza A virus suggests different roles for IgNAR and IgM, but a common immunodominance hierarchy with hemagglutinin on top
Authors: Altman Meghan O , Angeletti Davide , Yewdell Jonathon W , Flajnik Martin F
Published: January 2023

Cartilaginous fishes are the earliest extant phylogenic group to harness germline recombination and somatic hypermutation of immunoglobulins antibodies (Igs) for immunity. In this study we immunized and 1-year-later boosted, two adult nurse sharks (Ginglymostoma cirratum) with inactivated H1 influenza A virus (IAV). After immunization, polyclonal sera from these sharks had strong antiviral properties. Primary immune and recall sera neutralized viral growth in cell culture, caused hemagglutination inhibition, and inhibited neuraminidase (NA) activity. By ELISA and protein microarray, we examined the contribution of two shark Ig isotypes, IgNAR and IgM. Unique to cartilaginous fishes, IgNAR are heavy-chain-only Igs. IgNAR from immunized sharks bound only the immunizing strain of virus. But, flow cytometry of spleen and blood revealed ~1% of IgNAR-positive cells bound both H1 and H5 HA subtypes. While these crossreactive cells exist, any crossreactive antibodies made by cells in these lineages were below our limit of detection. By contrast, both preimmune and immune serum IgM was highly crossreactive, binding H1, H3, H5, and even headless HA. In cartilaginous fish, secreted IgM comes in two independently-produced forms high affinity monomeric (7s) and pentameric (19s). Serum fractionation determined these crossreactive IgM antibodies were 19s. While capable of binding virus, we found 19s alone could not neutralize virus. Despite these differences, regardless of isotype, and in-line with our previous work with mice and lamprey, the immunodominance hierarchy of shark vaccine-induced IgNAR and IgM antibodies to IAV is consistent—HA is dominant, followed by NA, and then core proteins, such as, nucleoprotein and matrix.
Infection with non-CD4 tropic variant of SIVmac239 accelerates the formation of mature germinal center reactions and elicits antigen specific B cell responses
Authors: Dimopoulos Yiannis Petros , Padhan Kartika , Moysi Eirini , Swanstrom Adrienne , Lifson Jeffrey D , LaBranche Celia , Blaire Robert , Aye Pyone , Schiro Faith , Lackner Andrew , Koup Richard , Petrovas Constantinos , Hoxie Jim
Published: January 2023

Background

What determines the development of germinal centers (GCs) in lymph nodes (LNs) early after HIV/SIV infection is likely to be crucial for developing neutralizing antibodies and B cell memory.

Materials and Methods

Rhesus macaques were inoculated i.v. with either SIVmac239 (n=2) or iMac239ΔD385 (iMacΔD) (n=4), a CD4-independent variant of SIVmac239 lacking a CD4 binding site (J. Virol. 90:4966, 2016). LNs from different time points were analyzed using polychromatic flow cytometry and multiplexed confocal imaging.

Results

In contrast to SIVmac239, iMacΔD infection was associated with relative preservation of total and CD28hiCD95hi memory CD4 T cells, consistent with its compromised capacity to target CD4 T cells. Imaging analysis revealed accelerated formation of mature, polarized GCs in iMacΔD compared to SIVmac239 infected animals, a profile associated with i) increased frequencies of PD-1hiBcl-6hi CD4 T cells, ii) higher levels of IL-10 and IL-21 within the follicles, iii) increased frequency of PH2AX+ (a required factor for isotype switching) B cells, iv) lower levels of Fas-L in the follicular areas, associated with lower TUNEL (cell death) positivity and v) lower accumulation of CD163+ cells (monocytes/macrophages; a surrogate of local inflammation) in proximity to follicles. The observed GC dynamics associate with earlier virus-specific antibody responses in iMacΔD compared to SIVmac239.

Conclusions

Our data indicate that CD4 tropism of SIV greatly affects the development of GC reactions early after infection. The iMacΔD model could provide critical information on how GC reactions can be manipulated to augment the generation of neutralizing antibodies and B cell memory.
Th17 cells confer both systemic and mucosal protection against Trypanosoma cruzi infection
Authors: Cai Catherine Wen , Blase Jennifer R , Eickhoff Christopher S , Hoft Daniel F
Published: January 2023

Chagas disease is caused by chronic infection with the Trypanosoma cruzi parasite. It affects 11 million people, a third of whom will develop life-threatening cardiac and gastrointestinal morbidities. Optimal immunity to this intracellular pathogen requires CD8+ and CD4+ T cell responses. Recently, we demonstrated that CD4+ Th17 cells can confer even stronger protection against T. cruzi infection than CD4+ Th1 cells, typically considered most important for intracellular immunity.

To study systemic protection, we adoptively transferred RAG KO mice with naive polyclonal CD8+ T cells and parasite-specific Th1 or Th17 cells, then infected them subcutaneously. Th17 cell co-transfer resulted in 100% long-term survival, compared to 38% with Th1 co-transfer. Mechanistically, the Th17 cells produced high levels of IL-21, which provided help for the activation of CD8+ T cells. Th17 cells given with IL-21R KO CD8+ T cells failed to protect mice, underscoring the importance of IL-21 signaling.

Although CD8+ T cells were required for systemic immunity, Th17 cells alone protected against mucosal infections. Mice reconstituted with Th17 cells prior to orogastric challenge had decreased parasite burdens in the stomach and lower levels of dissemination to other organs, indicating a direct protective effect. In vitro, we discovered that Th17 cells can directly suppress infection in macrophages via the IL-17A-dependent triggering of NADPH oxidase, important for the phagocyte respiratory burst response.

In summary, we have shown that Th17 cells drive robust systemic and mucosal immune responses against an intracellular parasite through both direct and indirect means, highlighting important new roles for Th17 cells in intracellular immunity.
Itk: A potential target for Saccharopolyspora rectivirgula-induced hypersensitivity pneumonitis
Authors: Koylass Nicholas Robert , Huang Weishan , Carter Chavez , Elmore Jessica , August Avery
Published: January 2023

Hypersensitivity pneumonitis (HP) is a lung disease caused by repeated inhalation of environmental antigens leading to inflammation, tissue scarring, and impaired lung function. This pathology is associated with IL-17A-producing Th17 cells and recruitment of inflammatory cells such as neutrophils to the lungs; on the other hand, Foxp3+ regulatory T (Treg) cells play a protective rolein HP by suppressing pathogenic T cell functions. The thermophile Saccharopolyspora rectivirgula (SR) causes HP in humans, attributed to the rare farmers’ lung disease. We used a murine model carrying the IL-17AGFP/Foxp3RFP dual reporters in SR-induced HP, to study the cellular and molecular mechanisms of HP disease development. IL-2-inducible T cell Kinase, ITK is downstream of the T cell receptor, and is required for effective differentiation of Th17 cells, but limits the expansion of Treg cells. In this work, we sought to determine the possibility of using ITK as a target for pharmaceutical inhibitors in HP. To evaluate the effect of targeting ITK kinase activity in HP therapy, we utilized an Itk allele sensitive mutant (Itkas) that allows specific inhibition by a compound 3MBPP1. WT, Itk−/− and ItkasIL-17AGFP/Foxp3RFP mice exposed to SR were treated with 3MBPP and analyzed for histopathology and various immune cell populations. Intranasal delivery of 3MBPP1 for up to 4 weeks affected pathogenesis and targeted primarily the bronchoalveolarlavage (BAL) immune subsets, including CD4+ IL-17A+ cells. In addition, Treg cells in the pulmonary lymph nodes, neutrophils in the lungs, and γδ T cells in all organs were affected by the treatment. These data suggest that ITK may be a therapeutic target for HP that involves the IL-17 and Foxp3-expressing T cell subsets.
Innate lymphoid cells (ILCs) from septic patients produce higher levels of cytokines than ILCs from healthy controls
Authors: Cruz D David , Niembro-Guerrero Ana Marcela , Ruiz-Sanchez Bibiana Patricia , Arriaga-Pizano Lourdes , Ferat-Osorio Eduardo , Chacon-Salinas Rommel , Estrada-Garcia Iris , Wong-Baeza Isabel
Published: January 2023

Sepsis is a systemic inflammatory response to infection, which leads to life-threatening organ dysfunction. During sepsis, many cells of the innate and adaptive immune systems have functional alterations; in particular, monocytes produce reduced levels of pro-inflammatory cytokines in response to microbial products (MAMPs). Innate lymphoid cells (ILCs) are cells with lymphocyte morphology that lack antigen-specific receptors and respond directly to cytokines and MAMPs; it is not known if ILCs are affected during sepsis. In this study, we used flow cytometry to evaluate the frequency of ILCs in the peripheral blood of septic patients and healthy controls, as well as the expression of CD56, CCR6 and NKp44 and the production of IL-17 and IFN-γ by ILCs. ILCs were defined as CD45+ lineage- CD127+ cells.

The number of total lymphocytes and the number of ILCs were significantly lower in septic patients, compared to healthy controls, with ILCs representing around 0.5% of peripheral blood lymphocytes. In both healthy controls and septic patients, CD56 defines two populations of ILCs: a higher percentage of the CD56− cells express CCR6, compared to the CD56+ cells, while a higher percentage of the CD56+ cells express NKp44, compared to the CD56− cells. CD56− ILCs are the main producers of IL-17, while CD56+ cells are the main producers of IFN-γ, in both healthy controls and septic patients. The production of these two cytokines is increased in the ILCs from septic patients, compared to the ILCs from healthy controls. This is in contrast with the decreased cytokine production that is observed in monocytes and T lymphocytes from septic patients.

This project was funded by CONACyT-México (219661).
Involvement of IL-17A-producing TCR γδ T cells in late protective immunity against pulmonary Mycobacterium tuberculosis infection
Authors: Umemura Masayuki , Okamoto-Yoshida Yuko , Yahagi Ayano , Nakae Susumu , Iwakura Yoichiro , Takaesu Giichi , Matsuzaki Goro
Published: January 2023

Interleukin (IL)-17A is a cytokine originally reported to induce neutrophil-mediated inflammation and anti-microbial activity. The CD4+ T cells which produce IL-17A have been well characterized as Th17 cells. On the other hand, IL-17A-producing TCR γδ+ T cells have been reported to participate in the immune response at an early stage of infection with Listeria monocytogenes and Mycobacterium bovis in mice. However, the involvement of IL-17A in protective immunity was not clearly demonstrated in the chronic stage of M. tuberculosis-infected mice. In this study, we found that TCR γδ+ T cells are a primary source of IL-17A, but that mycobacterial antigen-specific Th17 cells were hardly detected even at the chronic stage of M. tuberculosis infection. IL-17A-deficient mice showed a decreased survival rate, and increased bacterial burden in the lungs after the infection when compared to the wild-type mice. Furthermore, a histological analysis showed an impaired granuloma formation in the infected lungs of IL-17A-deficient mice, which was considered to be due to a decrease of IFN-γ and TNF-α at the chronic stage. These data suggest that the IL-17A-producing TCR γδ+ T cells, rather than the Th17 cells, in the infected lungs are an indispensable source of protective immunity against M. tuberculosis infection.
The role of IL-10 during early M. tuberculosis infection in a non-human primate model
Authors: Wong Eileen A , Kraus Carolyn , Reimann Keith A , Flynn JoAnne L
Published: January 2023

Tuberculosis (TB), caused by M. tuberculosis (Mtb), continues to be a global health problem. Lung granulomas, organized structures of host immune cells to contain Mtb, are the pathologic hallmark of TB. T cell cytokines play a major role in containment of Mtb infection within these granulomas. While the importance of pro-inflammatory cytokines IFN-γ and TNF in controlling Mtb infections has been established, the effects of immunomodulatory cytokines, such as IL-10, in Mtb infections are less well understood. We used cynomolgus macaques, a non-human primate (NHP) model that recapitulates human TB to investigate the role of IL-10 early in Mtb infection. Preliminary ex vivo studies of excised lung granulomas from Mtb-infected NHPs suggested IL-10 may be downregulating IL-2 cytokine production in TB lung granulomas. To further examine the role of IL-10, a cross-reactive, rhesus recombinant anti-IL-10 antibody was used to neutralize IL-10 in vivo in NHPs prior to and during the course of Mtb infection. Peripheral cytokine responses in the presence of IL-10 neutralization were analyzed serially by flow cytometry of peripheral blood mononuclear cells. Disease progression was monitored by PET-CT scans, and local immune responses in the granulomas assessed at necropsy. Our data indicate transient differences in inflammation early in infection between anti-IL-10-treated and control monkeys. Surprisingly, there was less inflammation in granulomas from IL-10 neutralized animals at 3–4 weeks post-infection, compared to control animals. Overall, this unique dataset provides important insight into the contribution of IL-10 to the necessary immunological balance for controlled bacterial burden and disease pathology in Mtb infections.
Role of p38 MAPK and NF-κB signaling in chromatin decondensation and acquisition of cytokine competence during T cell activation
Authors: Rawlings Jason S , Funsten Jen , Brizuela Keny Murillo , Bingham Kellie , Lee Megan , Mitchell Taylor , Meredith Jenna
Published: January 2023

A proper immune response is hallmarked by the activation and clonal proliferation of antigen specific T cells. Antigen presentation to the T cell receptor (TCR) leads to the production of two signaling molecules, IP3 and DAG. While IP3 induces calcium flux, DAG activates Protein Kinase C (PKC). The activation of these pathways results in the decondensation of chromatin and the production of interleukin-2 (IL-2), both of which are required for the clonal proliferation of activated T cells. Previous work in our lab demonstrated that the IP3 pathway is sufficient for chromatin decondensation. In this study, we show that this pathway is not sufficient to make cells respond to IL-2. Furthermore, we investigate the role of the DAG pathway, specifically the role of the downstream molecules p38 MAPK and NF-κB, in the decondensation of chromatin and the competence to respond to IL-2 signaling. We found that neither of these molecules play a role in chromatin decondensation and that NF-κB may play a role in IL-2 competence. Collectively, these data, along with our previous findings, suggest that chromatin decondensation and competence to respond to IL-2 may be controlled by IP3 and DAG signaling respectively.
IRF4 controls a core regulatory circuit of T cell dysfunction in transplantation
Authors: Wu Jie , Shi Xiaomin , Xiao Xiang , Minze Laurie , Wang Jin , Ghobrial Rafik M. , Xia Jiahong , Sciammas Roger , Li Xian C. , Chen Wenhao
Published: January 2023

T cell dysfunction has emerged as a key event leading to failure in the control of persistent infections and tumors. However, the transcriptional determinants for T cell dysfunction remain unknown. Here we show that IRF4 is a key transcriptional determinant of T cell dysfunction by restraining the dysfunctional differentiation process.

We use a heart transplantation model to decipher the molecular basis of T cell dysfunction. Heart allografts survived indefinitely in Irf4-deficient mice. This stable engraftment is due to the progressive establishment of effector T cell dysfunction. Mechanistically, IRF4 represses PD-1, Helios, and other molecules associated with T cell dysfunction. In particular, IRF4 has a profound impact on epigenetic accessibility of PD-1 cis-regulatory elements. In the absence of IRF4, chromatin accessibility as well as binding of Helios at PD-1 cis-regulatory elements are markedly increased in effector T cells, resulting in enhanced expression of PD-1 and T cell dysfunction. Intriguingly, although the dysfunctional state of Irf4-deficient T cells is initially reversible by blockade of PD-1/PD-L1 pathway, it progressively evolves into a “terminal” irreversible state within 30 days post-transplant. Lastly, we revealed that the MEK1/2 inhibitor, trametinib, dramatically decreases IRF4 expression in T cells, abrogates EAE development, and prolongs heart allograft survival.

Our results reveal a previously unappreciated aspect of T cell dysfunction, namely the repression of the dysfunctional differentiation of T cells by IRF4. Central control of T cell dysfunction by IRF4 has a significant potential impact on the course of future work in modulating T cell dysfunction
Celecoxib down-regulates Angiogenic factor of Retinal Pigment Epithelial cells in Hypoxic state via HIF-1α degradation.
Authors: KIM SEONHYUN , KIM JAYOUNG , HONG SEUNGWOO , KIM YEONGSEOK , HUR DAEYOUNG
Published: January 2023

Age-related macular degeneration (AMD) is a common disease causing blindness in the aging population. AMD affects central regions of the retina and choroid that can lose vision. This disease starts with drusen between retinal pigment epithelium(RPE) and Bruch’s membrane and finally results in neovascularization and destruction of retina. Hypoxic state is deeply associated with abnormal vessel formation in AMD. Hypoxia accumulate hypoxia-inducible factor-α(HIF-1α) and induce angiogenesis. In this study, we investigated that HIF-1α was upregulated in ARPE-19 cells which was exposed hypoxic states. Interestingly, celecoxib, an inhibitor of cyclooxygenase-2 (COX-2) could degrade HIF-1α on hypoxic RPE cells. We also observed that celecoxib blocked vascular endothelial growth factor (VEGF) production in hypoxic ARPE-19 cells, and conditioned media of celecoxib-treated ARPE-19 cells inhibited tube formations of HUVECs. We investigated that celecoxib might regulate degradation by ubiquitination/proteasome system, which were supported by MG-132, PYR-41 pretreatment experiments. Taken together, we suggest that these effects of celecoxib down-regulate angiogenesis through HIF-1α degradation and can support prevention of neovascularization of AMD.
The Nrf2 activators tBHQ and CDDO-Im have both Nrf2-dependent and – independent effects on human Jurkat T cell activation, as determined by CRISPR-Cas9 gene editing
Authors: Zagorski Joseph Ward , Maser Tyler P , Liby Karen T , Rockwell Cheryl Elizabeth
Published: January 2023

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a stress activated transcription factor, activated by such stimuli as reactive oxygen and electrophilic stress. Upon activation Nrf2 accumulates in the nucleus and activates a battery of target genes, many of which are cytoprotective in function. We have previously shown that a common synthetic food additive, tBHQ, is a potent Nrf2 activator and suppresses certain events of human T cell activation, namely, IL-2 secretion, CD25 expression, and transactivation ability of NFkB. The purpose of our current studies was to develop a Nrf2-null human model, in which it can be determined whether the effects of tBHQ upon the early events of T cell activation are Nrf2-dependent. Treatment of both Wild-type and Nrf2-null Jurkat T cells revealed that the suppression of IL-2 and NFkB by tBHQ is largely Nrf2-independent, whereas the suppression of CD25 is partially dependent upon Nrf2. Although interesting, these studies left the question as to the role of Nrf2 in T cell activation largely unanswered. Therefore, we utilized a more potent and selective Nrf2 activator, CDDO-Im. CDDO-Im treatment resulted in a Nrf2-dependent suppression of IL-2 secretion and an inhibition of NFkB activity that was partially Nrf2-dependent. Taken together, our model is the first to show that activation of Nrf2 suppresses early events of human T cell activation (This work is funded by NIH grants ES018885, ES024966 and GM092715).
Determining Fas Signaling Pathways by Quantifying Receptor Clustering
Authors: Frazzette Nicholas , Cruz Anthony , Sengupta Prabuddha , Lippincott-Schwartz Jennifer , Siegel Richard M.
Published: January 2023

The TNF-cytokine family receptor Fas (CD95) mediates T cell apoptosis by recruiting a death-inducing signaling complex (DISC) composed of the adaptor protein (FADD) and Caspase-8. Recently, it has been shown that Fas is also directs non-death signaling and enhances effector cell differentiation in T cells. Mutations which reduce the ability of Fas to localize to lipid raft microdomains and cluster upon activation abrogate the death-inducing potential of Fas while preserving non-apoptotic signaling. Caspase-8, a key mediator of both apoptotic and non-apoptotic signal, has been shown to form filamentous structures in vitro and upon overexpression in mammalian cells, but it is unknown if this process occurs in vivo to facilitate signaling. To investigate these supramolecular phenomena, we developed Fas and Caspase-8 fused to photoactivatable fluorescent proteins to be expressed in both primary T cells and T cell lines for super resolution imaging. Additionally, we have produced supported planar lipid bilayers presenting Fas ligand (FasL) and T cell stimulatory ligands to mimic a physiologic signaling environment. Photoactivatable localization microscopy (PALM) is used to observe and quantify clustering Fas molecules and formation of caspase-8 containing signaling complexes at nanometer scale resolution. Structure illuminated microscopy (SIM) and PALM will be used to observe the behavior of Fas relative to cellular-scale actin remodeling and TCR clustering. Our findings will reveal the behavior of supramolecular complexes mediating apoptosis and non-apoptotic signaling by Fas in the plasma membrane of living cells.
Alterations in B cell compartment induced by aging and impact of HIV infection.
Authors: Rinaldi Stefano , Pallikkuth Suresh , George Varghese , de Armas Lesley , Pahwa Rajendra , Sanchez Celeste , Pallin Maria , Pan Li , Cotugno Nicola , Dickinson Gordon , Rodriguez Allan , Fischl Margaret , Alcaide Maria , Gonzalez Louis , Palma Paolo , Pahwa Savita
Published: January 2023

Perturbations in B cells occur in both, chronological aging and in HIV infection. We investigated B cells in young (Y, <40yr), middle (M, 40–59yr) and old (O, ≥60yr) influenza vaccinated participants with controlled HIV infection (HIV, n=64) and HIV negative healthy controls (HC, n=60) at pre-vaccination (T0) and post-vaccination on d7 (T1) and d21 (T2). Results of B cell immunophenotyping of T0 PBMC were correlated against measures of vaccine responsiveness (hemagglutination inhibition (HAI) titers and antibody secreting cells in ELISpot) and age. Both HC and HIV demonstrated a negative correlation of age and Flu-specific HAI titer at T2 (p=0.005, and 0.019, respectively). However only HC demonstrated negative relationships of age with spontaneous ELISpot for plasmablast function at T1 (p=0.0001) and memory B cell ELISpot at T2 (p=0.05). Immunophenotyping of B cells in HC demonstrated increased double negative (DN) B cells and decreased plasmablasts in O compared to Y. In HIV however phenotypic and functional impairment of B cells were already evident in Y such that Y HIV were different from Y HC with increased DN B cells and decreased B ELISpot responses, while O HIV were similar to O HC. Additionally, B cells from Y HIV had increased frequencies of CD80 and PDL1 markers suggesting a heightened state of immune activation and exhaustion. Our results suggest that despite virological suppression, young HIV-infected individuals are similar to Old HC because of early impairment of B cells due to ongoing immune activation. Clinical implications are that vaccination strategies used for old HC may need to be implemented at an earlier age for HIV infected population for improved vaccine efficacy.
Multifunctional cytokine and T cell memory phenotype profiles differ in active versus cured tuberculosis.
Authors: Leong Samantha , Rodrigues Rodrigo Ribeiro , Ellner Jerrold J. , Dietze Reynaldo , Jones-López Edward C. , Alland David , Salgame Padmini
Published: January 2023

Tuberculosis, one of the most prevalent and deadliest infectious diseases worldwide, requires long-term multidrug treatment regimens for curing active clinical disease. However, a diagnostic method for identifying successful cure from TB disease following anti-TB chemotherapy remains elusive. The overall goal of this study was to assess differential patterns of cytokine production and memory T cell phenotypes to distinguish between active TB subjects and remotely cured TB individuals. Using peripheral blood mononuclear cells from subjects enrolled in a TB household contact study established in Vitória, Brazil, cells were stimulated ex vivo with TB antigens and evaluated using multi-parametric flow cytometry. Total cytokine and multifunctional cytokine analyses revealed an increase in the proportion of IL-17 producing CD4+ T cells in active TB subjects, while cured TB subjects showed a clear dominance of cells producing IFNγ and IL-2 or TNFα. Comparison of memory phenotypes revealed differences between proportions of several CD4+ T cell memory subsets, with an effector memory dominance in cured TB compared to an increased stem cell memory representation in active TB. Increased expression of exhaustion marker PD-1 in advanced stages of memory cell differentiation suggested its possible use as a surrogate indicator of memory subset fluxes, with possible application as a biomarker for distinguishing differences between cured TB and active TB individuals.
Natural H7N9 infection in humans boosts cross-group stalk-specific antibody responses broadly cross-reactive to heterosubtypic influenza hemagglutinins from both group 1 and group 2
Authors: Liu Lu , Nachbagauer Raffael , Zhu Lingyan , Huang Yang , Xie Xinci , Jin Shan , Zhang Anli , Wan Yanmin , Hirsh Ariana , Tian Di , Shi Xiaolin , Dong Zhaoguang , Yuan Songhua , Hu Yunwen , Krammer Florian , Zhang Xiaoyan , Xu Jianqing
Published: January 2023

Background

The outbreak of novel avian H7N9 influenza virus infections in China in 2013 has demonstrated the continuing threat posed by zoonotic pathogens. To decipher the responses during natural infection will enlighten the vaccine development.

Methods

We assessed the induction of heterosubtypic cross-reactive antibodies induced by H7N9 infection against a large panel of recombinant hemagglutinins and neuraminidases by quantitative ELISA, and novel chimeric hemagglutinin constructs were employed to dissect the anti-stalk or head humoral response.

Results

H7N9 infection induced strong antibody responses against divergent H7 hemagglutinins. Interestingly, we also found the induction of antibodies against heterosubtypic hemagglutinins from both group 1 and group 2 and a boost in heterosubtypic neutralizing activity but not in hemagglutination inhibitory activity. Kinetic monitoring revealed that heterosubtypic binding/neutralizing antibody responses typically appeared and peaked earlier than intrasubtypic responses as stalk-specific memory responses.

Conclusions

Our results indicate cross-group binding and neutralizing antibody responses primarily targeting at stalk region could be elicited after influenza natural infection and probably with proper immunogen in the presence of preexisting responses. These data support our understanding of the breadth of the post-infection immune response that could inform the design of future, broadly protective influenza virus vaccines.
Terminally differentiated CD8 effector T cells have NK-like features and are potent mediators of HIV-specific ADCC
Authors: Lal Kerri G , Naluyima Prossy , Costanzo Margaret C , Kijak Gustavo H , Eller Leigh Anne , Creegan Matthew , Michael Nelson L , Robb Merlin L , Bolton Diane L , Sandberg Johan K , Eller Michael A.
Published: January 2023

Chronic viral infection and persistent antigenic stimulation is associated with a rise of late-stage differentiated CD8 T cells including a unique subset of CD8 T cells expressing FCg receptor IIIa (CD16). Expansion of CD16+ CD8 T cells is observed in other chronic diseases like HCV, but not HIV-1 infection. CD16 mediates antibody dependent cellular cytotoxicity (ADCC), most notably on NK cells, and is associated with protection from HIV-1 acquisition. Using cryopreserved PBMC from healthy and HIV-1 chronically infected donors from Uganda, we compared phenotypic, transcriptional, and functional changes in CD16+ CD8 T cells. We found that chronic, untreated HIV-1 infection is associated with elevated numbers of CD45RA+CD57+ terminal effector CD16+ CD8 T cells compared to healthy individuals, and display a distinct transcriptional profile in between conventional CD8 T cells and NK cells, characterized by high IKZF2 and KLRF1, and low expression of IL7R. This transcriptional profile translates into a NKp80+ IL-7Rα-surface phenotype and high expression of the Helios transcription factor. Additionally, CD16+ CD8 T cells are capable of mediating ADCC at levels similar to that of NK cells. These CD16+ CD8 T cells are highly activated and correlate positively with expansion of the CD8 T cell compartment and plasma levels of soluble factors of antiviral immunity and inflammation, such as IP-10, TNF, IL-6, and TNFRII. These data indicate that terminally differentiated, effector CD8 T cells acquire innate, NK cell-like characteristics during chronic HIV-1 infection, and suggest that HIV-specific ADCC is part of the repertoire of functions CD8 T cells use to combat the ability of HIV-1 to escape the TCR-specific, anti-viral effects of CD8 T cells.
“TCR transgenic, gp100 reactive T cells to target lymphangioleiomyomatosis”
Authors: Ankney Christian Mathew , Le Poole I. Caroline , Dilling Daniel F. , Eby Jonathan M.
Published: January 2023

Lymphangioleiomyomatosis (LAM) is a rare lung disease presenting with benign tumors, affecting premenopausal women. LAM is diagnosed by HMB45 staining. We questioned whether gp100 expression by lesional cells lends opportunities to treat LAM by adoptive T cell transfer. As LAM tumors are sparsely infiltrated by T cells, we looked to melanoma and vitiligo as sources of gp100-reactive TCRs. The gp100-reactive TCRs T4H2, R6C12 and SILv44 were cloned into retro-and lentiviral vectors and introduced into primary human CD8+ T cells. Transgenic T cells were combined with melanoma cells, melanocytes and LAM cells and responses were measured by ELISA detecting IFN-γ, IL-17, TNF or IL-4 after 48 hrs. Cytotoxicity was measured by 51Cr release and CD107a expression, and in vivo using SCID/Beige mice challenged with 106 888A2 human melanoma cells and treated twice with 106 SILv44 transduced T cells. Transgenic T cells conferred specific reactivity to HLA-A2+, gp100+ cells, including human LAM10224 cells, in vitro. The SILv44 TCR mediated most IL-17a expression whereas INF-γ secretion was greatest in T4H2 transduced cells. Surprisingly, IL17a expression decreased with increasing peptide concentrations. IL17a+, SILv44+ T cells conferred most anti-tumor protection in mice. LAM10224 cells can form tumors in immunodeficient mice, and SILv44 transgenic T cells should likewise contain such tumors. Thus, SILv44 transgenic T cells may serve as a viable treatment for LAM.
CCL3 augment antitumor immune priming in the tumor draining lymph node
Authors: Allen Frederick , Rauhe Peter , Askew David , Eid Saada , Nthale Joseph , Myers Jay , Tong Alexander , Huang Alex Yee-Chen
Published: January 2023

Lymph nodes (LNs) represent an area of interest for targeting metastatic tumors because they play an essential role in tumor survival in non-native tissues. The chemokine CCL3 is important in recruiting first-responder immune cells to areas of epithelial insult and orchestrating cellular contacts in the LNs of vaccinated mice that subsequently lead toward the enhancement of memory CD8+ T cell generation. We hypothesize that by introducing a continuous supply of CCL3 into the microenvironment of a metastatic tumor, we can redirect a LN destined for tumor-tolerance toward the production of greater antitumor cellular responses. To interrogate our hypothesis, we subcutaneously inoculated naïve murine recipients with a Balb/c colon metastatic tumor (CT26) that is either the wild-type (WTTUs) or WTTUs transfected to secrete CCL3 (L3TUs). Immunocompetent mice injected with L3TUs resulted in a suppression of tumor growth compared to the WTTU. In vivo analysis across 7-days post-tumor injections with WTTUs or L3TUs, revealed an enhanced accumulation of endogenous DCs, NKs, and lymphocytes in the TDLNs. In vitro analysis showed that OVA-peptide or whole-protein-pulsed bone marrow derived dendritic cells (BMDCs) cultured with CCL3, showed an enhanced capacity to induce proliferation of antigen-specific T cells. Examination of the day-5 TDLNs for direct signs of adaptive antitumor responses also revealed an enhanced production of the antitumor cytokine, IFNγ compared to WTTUs and the specific recruitment of antitumor-associated DCs to the TDLN.
IRF3 contributes to IFN-γ expression during B16F10 tumor growth and also IFN-γ-dependent reduction in B16 tumor cell growth
Authors: Petro Thomas M , Brown Deborah M , Guinn Zachary
Published: January 2023

Previously, we reported in C57Bl/6 (B6) and IRF3KO mice that Interferon Regulatory Factor (IRF-3) contributes to control of B16 melanoma tumor growth. Moreover we found a link between IRF-3 and expression of IFN-γ during in vitro T cell responses of B6 and IRF3KO mice. To investigate IRF3 and IFN-γ expression at growing tumors, we injected B6 and IRF3KO mice sq with B16-F10 tumor cells that constitutively express luciferase. Tumor growth was similar between B6 and IRF3KO mice at day 2 and 6, but was significantly greater at day 9 in IRF3KO mice compared with B6 mice. Using transcription factor assays of spleen extracts, IRF3 and IRF7 activation peaked at day 6 in B6 tumor-bearing mice but failed to occur in IRF3KO mice post injection. Likewise, significant induction of IFN-γ occurred in spleens and tumors in tumor-bearing B6 mice but failed to occur in tumor-bearing IRF3KO mice from day 6–9 post injection. Previous reports from other labs showed that the anti-tumor properties of IFN-γ are the result of G1/G0 cell cycle arrest. Using B16 cells or B16 cells deficient in IFN-γ receptor (B16-IRFGRKO), we found that IFN-γ decreases B16 tumor cell growth and synergizes with the TLR3/IRF3 agonists, poly I:C, in decreasing B16 cell growth. Concomitantly, IFN-γ induced interferon stimulated gene-54 (ISG54), which is IRF3 dependent and promotes apoptosis in cells. Moreover, we treated B16-SEAP and B16-SEAPIFNGRKO cells, which express secreted alkaline phosphatase (SEAP) under the control of an ISG54/ISRE promoter. We found that IFN-γ induces ISG54 promoter activity and poly I:C synergizes with IFN-γ to induce ISG54 in B16 cells. Thus an IFN-γ/IRF3/ISG54 nexus can significantly contribute to tumor cell control during anti-tumor immune responses.
GSK-J4 a potent and selective inhibitor of the H3K27 demethylase JMJD3/UTX limits acute colitis by de novo synthesis Retinoic Acid in dendritic cells
Authors: Rosemblatt Mario , Doñas Cristian , Neira Jocelyn , Osorio-Barrios Francisco , Carrasco Macarena , Pacheco Rodrigo , Loyola Alejandra
Published: January 2023

JMJD3 a demethylase of lysine 27 of histone H3 (H3K27) plays an important role in immune cells and inflammatory responses, and thus appears as an attractive target for the treatment of inflammatory diseases. Recently, GSK-J4, a selective and potent JMJD3 inhibitor was synthesized. We previously showed that GSK-J4 induced a tolerogenic phenotype on dendritic cells (DCs) promoting the generation and stability of induced Tregs as well as enhancing their suppressive function. However, the underlying mechanisms remain unclear.

Because GSK-J4 acts upon DCs, we evaluated RA synthesis as underlying pro-tolerogenic mechanisms affected by this drug.

Here we report that GSK-J4 treatment promoted RALDH activity on DCs and induced the expression of RALDH1 and RALDH3 enzymes in DCs, resulting in an increase in the expression of Foxp3, CCR9 and α4β7 in CD4+ T cells.

The increased RALDH1 and RALDH3 expression correlated with changes on the ratio of repressive (H3K27me3) versus active marks (H3K4me3) on RALDH1 and RALDH3 promoters in GSK-J4-treated DCs. Moreover, we demonstrated that systemic administration of GSK-J4 ameliorates the severity of DSS-induced acute colitis in mice.

Collectively this study demonstrates that GSK-J4 attenuates DSS-induced acute colitis, an effect that may be attributed to the de novo RA synthesis on DCs.

This drug may be a promising approach for the treatment of inflammatory diseases.
Amelioration of neuropsychiatric systemic lupus erythematosus by fingolimod-mediated sphingosine-1 phosphate receptor modulation.
Authors: Mike Elise V , Putterman Chaim
Published: January 2023

Approximately 40% of SLE patients suffer from neuropsychiatric manifestations, including cognitive dysfunction and depression. While the pathogenesis of neuropsychiatric lupus (NPSLE) is not fully understood, critical aspects of disease development include neuroinflammation and the loss of blood-brain barrier (BBB) integrity. Fingolimod is an FDA-approved sphingosine-1 phosphate (S1P) receptor modulator that has been shown to reduce CNS lymphocytic infiltration and prevent BBB disruption in multiple sclerosis. In this study, we investigated the effects of fingolimod in MRL-lpr/lpr mice, a classic spontaneous SLE model. Ten-week-old mice were treated prophylactically three times weekly with vehicle alone or fingolimod (3mg/kg, n=16/group). After 4 weeks of treatment, mice were assessed for alterations in cognitive function or affective behaviors.

Fingolimod-treated mice exhibited significantly improved preference for novel objects (p=0.019) and objects in novel placement (p=0.007) when compared with control-treated mice, thereby demonstrating maintenance of recognition memory and visual memory, respectively. Mice treated with fingolimod also displayed less despair-induced immobility during the forced swim test (p=0.017), which indicates that fingolimod may mitigate depression. Preliminary histological assessment suggests decreased perivascular and periventricular leakage of serum albumin in fingolimod-treated mice. A reduction in lymphocytic infiltration of the choroid plexus in the lateral ventricles was also observed.

Our results highlight the important role of S1P in the pathogenesis of neurocognitive manifestations of SLE and receptor modulation as a potential therapeutic target for NPSLE.