bluuebunny/crossref_metadata_2025 huggingface dataset:
The Lactobacillus rhamnosus R0011 secretome attenuates TNFα-induced pro-inflammatory gene expression in human HT-29 intestinal epithelial cells
Authors: Jeffrey Michael P , MacPherson Chad , Mathieu Olivier , Tompkins Thomas A , Green-Johnson Julia May
Published: January 2023

Probiotic lactic acid bacteria have been associated with a wide array of host-immune modulatory effects including modulation of pro-inflammatory gene expression. However, many questions remain about the exact cellular mechanisms through which these bacteria modulate host cell responses following pro-inflammatory challenge. The aim of this study was to elucidate the effects of the Lactobacillus rhamnosus R0011 secretome on TNFα-induced pro-inflammatory gene expression. Previous analysis has shown that the <10kDa fraction of the Lr R0011 secretome attenuates TNFα-induced IL-8 production from HT-29 intestinal epithelial cells (IECs). Whole-genome wide microarray analysis was used to further interrogate the impact of the <10kDa fraction of the Lr R0011 secretome on TNFα-induced gene expression. Contact with the <10kDa secretome fraction alone induced minimal changes in global gene expression by HT-29 IECs, with no increases in pro-inflammatory gene expression. However, the <10kDa fraction of the Lr R0011 secretome attenuated TNFα-induced expression of the pro-inflammatory mediators CCL10, CXCL1, CXCL10, CXCL11, IL-1β, IL-8, IL-17C, IL-23A, IL-32, and PTGS2, when compared to HT-29 IEC treated with TNFα alone. Co-challenge of HT-29 IECs with the <10kDa fraction of the Lr R0011 secretome and TNFα also resulted in an increase in the expression of dual specificity phosphatase 1 (DUSP1), a key regulator of the MAPK pathway, and of activating transcription factor 3 (ATF3), a negative regulator of innate immunity, relative to HT-29 IECs treated with TNFα alone. This provides insight into mechanisms through which these bacteria may influence innate immune activity at the IEC level through soluble mediators.
The cytosolic DNA- and RNA-sensing pathways play important and non-redundant roles in host defense against vaccinia infection
Authors: Deng Liang , Dai Peihong , Wang Weiyi , Santagostino Sara , Serna-Tamayo Cristian , Meyer Cindy , Wang Yi , Li Tuo , Chen Zhijian J , Colonna Marco , Tuschl Thomas , Merghoub Taha , Shuman Stewart
Published: January 2023

Vaccinia virus is a prototypic poxvirus, and during the life cycle of the virus, viral DNA and RNA can be detected by the nucleic acid-sensing pathways to induce the expression of type I IFN and IFN-stimulating genes, which leads to the establishment of antiviral state. Poxviruses have evolved multiple strategies to evade host immunity. Vaccinia E3, which consists of two distinct Z-DNA-binding and dsRNA-binding domains, is a critical virulence factor. Virus lacks E3L gene (ΔE3L) is non-pathogenic in wild-type mice in an intranasal infection model. We have previously reported that ΔE3L infection of murine primary keratinocytes (KCs) induces Ifnb, Il6, Ccl5 and Ccl4 gene expression and protein secretion via a MAVS/IRF3-dependent mechanism. Here we show that MDA5, a cytosolic dsRNA sensor, is essential for the induction of innate immunity by ΔE3L in KCs, whereas PKR, another cytosolic dsRNA sensor, is not. ΔE3L gains virulence in MDA5, MAVS, or IRF3-deficient mice, but not in STING-or MyD88-deficient mice, in an intranasal infection model. The MDA5-, MAVS-, or IRF3-deficient mice lost 20% of weight but all recovered in response to ΔE3L infection. By contrast, both the MDA5 and STING-mediated sensing pathways contribute to host defense against WT vaccinia virus infection. Lastly, whereas ΔE3L virus fails to replicate in wild-type murine primary fibroblasts, it gains replication capacity in cGAS or STING-deficient murine primary fibroblasts. Taken together, these results indicate that vaccinia viral nucleic acids are detected via the cytosolic DNA- and/or RNA-sensing pathways in a cell-type dependent manner, and both pathways play important and non-redundant roles in host defense against vaccinia infection.
Antiviral activity of cell free hypothiocyanite against various subtypes of Influenza virus
Authors: Gingerich Aaron , Patel Urmi , Hanson Jarod , Rada Balazs , Tripp Ralph A
Published: January 2023

The respiratory epithelium is one of the first lines of defense utilized by the body to fend off invading pathogens. One such defense mechanism is a three-component system made up of the thiocyanate ion (SCN−), lactoperoxidase (LPO) and hydrogen peroxide (H2O2) which together create antimicrobial hypothiocyanite ions. Cell derived H2O2 is produced by the NADPH oxidase, dual oxidase enzymes (DUOX1&2). This system is capable of functioning with halides such as iodide as a substitute for thiocyanate. Previously we were able to show that cell derived hypothiocyanite was capable of inactivating influenza virus with a 4–5 log efficiency. We used a cell free method utilizing glucose oxidase as our source of H2O2 to create a high throughput in vitro system to determine that hypothiocyanite inactivates Influenza in a strain independent manner and that thiocyanate is the key substrate in the reaction. We also determined that the more acidic the pH the more efficient the viral inactivation. Additionally, we substituted SCN− with I− and found that different substrates have different levels of antiviral activity. These results support the idea that in vivo this LPO/SCN/H2O2 system plays a vital role in the initial defense against influenza virus and that this system has the potential to change the course of an IAV infection.
Pulmonary monocytes promote the establishment of lung tissue-resident CD8 T cell memory following influenza infection
Authors: Dunbar Paul , Wein Alexander , Kumar Nivedah , Li Zheng-Rong , Cartwright Emily Kathleen , Hayward Sarah L , Kohlmeier Jacob E
Published: January 2023

Lung tissue resident memory CD8 T cells (TRM) are critical for protection against heterologous influenza virus challenge by promoting rapid viral clearance. Unlike other mucosal tissues, we have found that local environmental signals are insufficient for the establishment of lung TRM, and that pulmonary antigen encounter is necessary for TRM to differentiate in the lung. Therefore, we investigated the role of lung antigen presenting cell (APC) subsets in the establishment of lung CD8 TRM following influenza infection. The initial appearance of flu-specific CD8 T cells in the lung with a TRM phenotype begins around day 10 post-infection, which coincides with viral clearance. Correspondingly, while most lung APC subsets contain influenza antigens over the course of infection, monocytes are the most numerous lung APC subset containing influenza antigens at the time of TRM appearance. Furthermore, imaging shows direct interaction between monocytes and T cells in the lung, supporting the idea that monocytes can act as drivers of lung TRM differentiation. In a murine influenza model, CCR2−/− mice, which have a defect in monocyte recruitment to the lung, had significantly fewer flu-specific lung CD8 TRM than WT controls. Notably, this defect was not observed in the flu-specific CD8 memory T cells in the lung vasculature or mediastinal lymph node, indicating this effect is specific for tissue-resident memory cells. In addition, this phenotype is not driven by a lack of CCR2 on T cells, as adoptive transfer of wild-type OT-I T cells into CCR2−/− mice yields the same deficiency of lung TRM. Thus, these data show that antigen presentation by lung CCR2+ monocytes has an important role in the establishment of lung CD8 TRM.
Identification of antigen-specific, lung resident memory B cells after influenza infection
Authors: Allie S Rameeza , Bradley John E , Mudunuru Uma , Randall Troy D.
Published: January 2023

B cells display phenotypic and functional heterogeneity in multiple anatomical locations following vaccination or infection. Influenza-specific memory B cells (Flu+BMEM cells) are found in both lymphoid tissues and lung. It is unclear whether these cells represent circulating or resident memory B cell (BRM) populations. We hypothesized that a portion of the Flu+BMEM cell population in the lung would be non-circulating, BRMs. To determine whether Flu+BMEM cells in the lung are circulating or resident, we parabiotically joined previously-infected, congenically-mismatched mice for 2 weeks and marked those cells currently in circulation by infusing anti-B220 and identified those that have trafficked between the partners by their expression of the CD45 congenic marker. The lungs of these mice had large populations of hemagglutinin and nucleoprotein -specific BMEM cells that did not attain equilibrium within 2 weeks of parabiosis, suggesting that they are non-circulating. The Flu+ BRMs in the lungs consisted of 56% IgM+ and 43.9% isotype-switched BRMs. They were established as early as 15 days after infection and maintained for at least 60 days. The formation of Flu+ BRMs required the germinal center (GC), as blocking CD40L with MR1 antibody, during the primary infection abrogated BRM. However, MR1-treatment of mice with established BRM did not affect BRMs in the lung, even though Flu+ GC B cells could be detected in the LN for up to 90 days. These data suggest that GC-dependent lung-BRMs are established early after infection and maintained independently of GCs. These findings are important in the development of vaccines that elicit BRMs and they will provide mechanistic information into the function of Ag+BMEM cells residing in the mucosa.
Staphylococcus Enterotoxin B mediates Acute Lung Injury through alterations in the composition of microbiome in Gut-Lung Axis
Authors: Sultan Muthanna Ali , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

Staphylococcus Enterotoxin B (SEB), produced by Staphylococcus aureus is a super antigen that triggers cytokine storm, and inhalation of SEB leads to toxic shock syndrome, respiratory failure, and often death. Microbiome plays an important role in the development and functions of immune system in different parts of the body Including respiratory and digestive systems. Also, cross talk between mucosal tissue of gut and lung through Gut Lung Axis (GLA) can influence immune and respiratory functions of the lungs. In the current study, we used intranasal exposure of SEB to induce acute lung injury (ALI) in C57BL/6 mice and performed 16S rRNA metagenomics sequencing to investigate nature of alterations in gut and lung microbiome after induction of ALI by SEB when compared to controls. Analysis of microbiome for Caecal flush and lung revealed that SEB inhalation caused a decrease in some species such as Ruminococcus gnavus in the lung and gut while increasing Akkremensia mucinphilia, which is responsible for degrading mucin barrier that separates commensal bacteria and pathogenic bacteria. We also found a decrease in some genus level in SEB group when compared to control group such as Lactobacillus. Collectively, these data suggest that exposure to SEB that triggers ALI, is associated with alterations in the composition of microbiome, which may regulate SEB-meadited pathogenesis in the lungs
Novel role for protein kinase D3 (PKD3) in airway epithelial activation and lung inflammation in response to double stranded RNA
Authors: Veazey Janelle , Chapman Timothy J. , Smyth Timothy , Hillman Sara , Georas Steve
Published: January 2023

Airway epithelial cells (AEC) are among the first to encounter inhaled particles and viruses and initiate subsequent immune responses. We recently reported that protein kinase D (PKD) regulates the barrier function of AEC in vitro after stimulation with the double stranded RNA polyI:C (PMID 21996340). Exposure to polyI:C mimics many features of respiratory viral infection in AEC, including induction of pro-inflammatory cytokines. It remains unknown whether PKD plays a role in AEC cytokine secretion, which we explored both in vitro and in vivo. First, we found that polyI:C-induced IL-8 production in 16HBE cells (a human AEC line) was markedly attenuated by pretreatment with PKD antagonist (CRT0066101 from Tocris (from 13000 ± 3752 to 3676 ± 2224 pg/ml at 5 μM CRT, n=4, p<0.05). Next, we challenged mice with inhaled polyI:C (10 μg, 3 consecutive days) and observed a significant increase in bronchoalveolar lavage (BAL) neutrophils 24 hours post challenge (from 0.4 ± 0.8 to 31.2 ± 9.4%; n=10, p<0.01). Strikingly, BAL neutrophil influx was reduced to near baseline values in mice pre-treated with CRT in a dose-dependent manner (e.g. 3.7 ± 4.7% with 100 μg CRT, n=10, p<0.01). The PKD antagonist also markedly reduced BAL levels of the neutrophil chemoattractant KC (p<0.01). PKD3 is the most abundant isoform in the lung by Western blot and RT-PCR. Finally, we found that polyI:C challenge of PKD3+/− mice (Prkd3Gt(OST191038)Lex/MMUCD from MMRRC) caused roughly 50% less BAL neutrophilia and KC release compared to age- and sex-matched wild type littermates (n=5). We conclude that PKD3 plays a previously unsuspected role in regulating AEC activation in response to double stranded RNA, suggesting a novel role for PKD3 in anti-viral immunity.
VSIG-3/IGSF11 is a ligand of VISTA/PD-1H and inhibits human T cell function
Authors: Wang Jinghua , Wu Guoping , Manick Brian , Hernandez Vida , Renelt Mark , Bi Ming , Li Jun , Kalabokis Vassilios
Published: January 2023

B7 family members and their receptors play a central role in the regulation of T cell responses through T cell costimulation and coinhibition pathways that constitute very attractive targets for the development of immunotherapeutic drugs. In this study, we report that VSIG-3/IGSF11 is a ligand of B7 family member VISTA/PD-1H and inhibits human T cell functions through a novel VSIG-3/VISTA pathway. Extensive functional ELISA binding screening assay reveals that VSIG-3 does bind the new B7 family member VISTA, but does not interact with other known members of the B7 family. Furthermore, VSIG-3 inhibits human T cell proliferation in the presence of T cell receptor signaling. In addition, VSIG-3 significantly reduces cytokine and chemokine production by human T cells including IFN-γ, IL-2, IL-17, CCL5/Rantes, CCL3/MIP-1alpha, and CXCL11/I-TAC. Anti-VISTA neutralization antibodies attenuate the binding of VSIG-3 and VISTA, as well as VSIG-3 induced T cell inhibition. Thus, we have identified a novel B7 pathway that is able to inhibit human T cell proliferation and cytokine production. This unique VSIG-3/VISTA coinhibitory pathway may provide new strategies for the treatment of human cancers, autoimmune disorders, infection, and transplant rejection and may help to design better vaccines.
Type 1 regulatory T cells (Tr1) homeostasis and function in aging
Authors: Almanan Maha A , Raynor Jana , Chougnet Claire , Salamonis Nathan , Amarachintha Surya , Steinbrecher Kris , Hildeman David A
Published: January 2023

Type 1 regulatory T cells (Tr1) are a unique population of CD4+ Foxp3− cells that express high levels of IL-10, and have been defined based on their expression of CD49b and LAG-3. Despite the critical roles played by Tr1 cells in controlling T cell responses in autoimmunity and infection, the mechanisms underlying the homeostasis of Tr1 cells remain unclear. Here, we investigated the homeostasis, phenotype and function of Tr1 cells with age. We found that Tr1 cells accumulated dramatically with age. Further, Tr1 cells produced more IL-10 per cell compared to Foxp3+ Treg. While aged Tr1 cells expressed significant levels of c-Maf and Egr-2, two of the Tr1 defining transcription factors, they largely lacked expression of CD49b and were heterogeneous in LAG-3 and ICOS expression. Despite this heterogeneity, LAG-3 identified a population of aged Tr1 cells that potently inhibited T-cell proliferation in a partially IL-10-dependent manner in vitro and controlled colitis severity in vivo. Additional flow cytometric analysis revealed that roughly half of the IL-10 producing cells co-produced IFN-γ. Further, single cell mRNA-seq analysis revealed that IL-10+IFN-γ− cells had a TGF-beta signature, while IL-10+IFN-γ+ cells had a STAT4 signature. Strikingly, IL-6 was critical for overall Tr1 cell accumulation, as aged IL-6 KO mice showed a profound reduction in Tr1 cells and their expression of c-Maf. Combined, these data show that IL-6 promotes Tr1 accumulation with age and provide a new insight into a novel feedback loop whereby an inflammatory cytokine drives accrual of an anti-inflammatory cell population.
PD-L1 binds with B7-1 only in cis on the same cell surface
Authors: Chaudhri Apoorvi , Xiao Yanping , Zhu Baogong , Freeman Gordon J.
Published: January 2023

Programmed death ligand 1 (PD-L1)-mediated immune suppression is a major regulator of peripheral tolerance and is often co-opted by tumors to evade immune attack. PD-L1 binds to PD-1 but in addition binds to B7-1 (CD80) to regulate T cell function. The binding interactions of the PD-L1-B7-1 pathway and its functional role need further investigation in order to understand differences between PD-1 and PD-L1 mediated tumor immunotherapy. We examined the molecular orientation for PD-L1 binding to B7-1 using cell to cell binding assays, ELISA, and flow cytometry. Surprisingly, we found that PD-L1 transfected cells did not bind to B7-1 transfected cells, whereas PD-L1 transfected cells bound to PD-1 transfected cells. As expected, B7-1 transfected cells bound to CD28 or cytotoxic T-lymphocyte antigen 4 (CTLA-4) transfected cells. By ELISA and flow cytometry with purified proteins, we found that PD-L1 and B7-1 had a strong binding interaction only when the PD-L1 molecule was accessible and flexible. On decreasing its accessibility, the binding was greatly reduced. We observed the PD-L1-B7-1 interaction competed for the binding of PD-L1 to PD-1. Further, we co-transfected PD-L1 and B7-1 on the same cell surface and confirmed the cis binding using a Nanobit Assay system. These results indicate that the PD-L1-B7-1 interaction can occur in cis when the molecules are on the same cell but not in trans when the molecules are on different cells. This binding orientation emphasizes the functional importance of co-expression of B7-1 and PD-L1 on the same cell. Interaction in cis competes for binding to other receptors and modulates their function. Our findings help in further understanding PD-L1 mediated immune suppression.
A15153G mAb, a novel tool for human TIGIT study
Authors: Li Cheng-Rui , Li Mong-Shang , Yang Xi-Feng
Published: January 2023

T cell immunoglobulin and ITIM domain receptor (TIGIT) is a recently identified member of the PVR (poliovirus receptor) family of immunoglobulin-like domain containing proteins that is expressed on subsets of T cells and NK cells. With mouse model studies, TIGIT has been established as a new co-inhibitory, or “checkpoint” molecule in immune regulation. It is thus considered as a potential therapeutic target for cancer and for autoimmune/inflammatory disorders. However, due to limited availability of antibody reagents to human TIGIT, the biological function of this molecule in human cells, as well as its detailed mechanisms, has not been fully elucidated. We have developed a novel monoclonal antibody, clone A15153G, specific to human TIGIT. As expected, this antibody reacts with a subset of human peripheral blood T cells and most CD56+ NK cells. When A15153G antibody was applied in culture, it strongly suppressed anti-CD3 induced human T cell proliferation, suggesting that A15153G is an agonistic antibody. By multi-color flow cytometric analysis using this antibody, we found that the expression patterns of human TIGIT on the surface of CD4+ and CD8+ T cells are different. In the CD4+ T cells, TIGIT is mainly expressed in memory-like population; in contrast, significant percentage of “naïve” CD8+ T cells also expressed high levels of TIGIT in addition to memory population. Moreover, CD8+ T cells generally express higher levels TIGIT than CD4+ T cells. Studies of cytokine production profiles of TIGIT expressing cells are ongoing.
Associations between inflammation and senescent/exhaustion markers, CD57, KLRG1, and PD-1 differ in elderly and young
Authors: Shive Carey , Banks Richard , Popkin Daniel , Bonomo Robert , Canaday David H , Anthony Donald
Published: January 2023

Background

Inflammatory indices such as IL-6 and soluble CD14 are elevated in the elderly, and aging is also associated with immune senescence and exhaustion. Soluble indices of inflammation and immune senescence have independently been associated with mortality and morbidity in the elderly. Whether these pathways are mechanistically linked is not clear.

Methods

This pilot study examined the relationship of plasma levels of IL-6, IP10, and sCD14 to T cell expression of immune checkpoint markers PD-1, CD57, and KLRG1 inelderly (≥70 years old; n=10) and young (≤40 years old;n=10) participants.

Results

Elderly participants had elevated plasma levels of IL-6 (p= <0.0001), sCD14 (p=0.0009), and IP10 (p= 0.012) compared to young participants. Surface expression of PD-1 (p= 0.002), CD57 (p= 0.0005), and KLRG1 (p= 0.04) was elevated on memory CD4 T cell in elderly participants. Co-expression of immune checkpoint markers PD-1, CD57, and KLRG1 on CD4 T cells inversely correlated with plasma levels of IL-6 in the elderly; whereas coexpression of checkpoint markers on CD8 T cells positively correlated with plasma levels of IP10 in young participants. Plasma levels of sCD14 positively associated with PD-1 expression on CD4 TEM cells in elderly but inversely correlated with PD-1 expression onCD8 CM and TEM cells in young participants.

Conclusion

The relationship between soluble indices of inflammation and immune check pointmarker expression varies between elderly and young and between CD4 and CD8 T cells. Further studies are warranted to understand the complex interactions of inflammation and immune senescence and how they contribute tomortality and morbidity in the elderly.
Natural nano particle MIR2911 suppresses tumor growth by targeting TGFβ pathway.
Authors: Zhou Zhen , Wang Lintao , Zen Ke
Published: January 2023

Malignant tumor is one of the major diseases threatening the health of human beings. In many human cancers, transforming growth factor beta (TGFβ) expression is increased. Further researches also indicate that, in the development of tumors, TGFβ promotes the formation and development of tumors through a variety of mechanisms. Our research group previously found that traditional Chinese medicine honeysuckle encodes a special miRNA, MIR2911, with high expression level and stability. MIR2911 can be absorbed through GI tract and inhibit many influenza virus subtypes replication. Our study found that MIR2911 is more stable than many other miRNAs. With up to 85% of the GC content, we hypothesized that several MIR2911 moleculars could combine with each other and form natural nanoparticles, which can easily enter into tumor cells. Bioinformatics analysis also showed that MIR2911 can target TGFβ and inhibit its expression. This project intends to use the natural nanoparticles formed by MIR2911 as a tumor therapeutic drug, by inhibiting the TGF pathway, to achieve the purpose of the treatment of tumor.
Constitutive expression of TIGIT defines a population of CD4+ regulatory T cells in Bcell non-Hodgkin lymphoma
Authors: Yang Zhi-Zhang , Kim Hyo Jin , Price-Troska Tammy , Jalali Shahrzad , Villasboas Jose C. , Novak Anne J. , Ansell Stephen M.
Published: January 2023

T cell Ig and ITIM domain (TIGIT) is an immune checkpoint molecule and its ligation delivers an inhibitory signal to T cells that negatively regulates anti-tumor responses. However, the expression and biological relevance of TIGIT in B-cell non-Hodgkin lymphoma (NHL) is completely unknown. To phenotypically characterize TIGIT+ T cell subsets, we profiled TIGIT-expressing CD4+ and CD8+ T cells from biopsy specimens of B-cell NHL. TIGIT expression was not detectable on T cells from peripheral blood, however, TIGIT is constitutively expressed on CD4+ T cells from biopsy specimens of B-cell NHL with a median of 44.8% (range: 25.9–62.2, n=8) of CD4+ T cells expressing TIGIT. By profiling the CD4+TIGIT+ T cells, we observed that CD25 was highly expressed on intratumoral CD4+TIGIT+ T cells. Intracellular staining revealed that Foxp3 was expressed by intratumoral CD4+TIGIT+ T cells. These intratumoral CD4+TIGIT+ T cells expressed high level of chemokine receptor CCR4 and were absent of IL-7 receptor-α (CD127). Functionally, CD4+TIGIT+ T cells displayed reduced cytokine production, as the number of IFN-γ- and TNF-α-producing cells was lower in the TIGIT+ population than in TIGIT− T cells. While TIGIT expression can be induced by TCR activation, treatment of T cells with IL-2 and TGF-β inhibited TCR-mediated TIGIT induction. Furthermore, lymphoma B cells were involved in TIGIT induction as in vitro depletion of lymphoma B cells altered upregulation of TIGIT on CD4+ T cells in B-cell NHL. Taken together, these results indicated that constitutive expression of TIGIT defines a CD4+ population with similar phenotype to regulatory T cells. Inhibition of TIGIT signaling may be an additional mechanism to prevent T-cell suppression in B-cell NHL
Targeting human acute myeloid leukemia with multi-epitope specific naïvederived anti-tumor CD4+ T cells
Authors: Chang Mark Jesse , Whitehill Gregory , Stegemann Scott , Scofield Eric , Wong Susan , Keyvanfar Keyvan , Jain Prachi , Ito Sawa , Chen Jichun , Barrett John , Muranski Pawel
Published: January 2023

Little is known about the anti-tumor potential of human CD4+ T cells. Hematological malignancies constitutively expressing MHC class II could be a target for tumor-specific CD4+ T cells. We studied antigen-specific human CD4+ T cells targeting leukemia associated antigens (LAA) WT-1, PRAME, MAGEA A3 and Ny-ESO1 in vitro and in a humanized NSG mouse acute myeloid leukemia (AML) model. We hypothesized that naïve CD4+ T cells are an important source of anti-tumor T cells. Bulk and naïve CD4+ T cells isolated from the blood of normal donors were stimulated with autologous dendritic cells pulsed with overlapping 15mer peptides spanning the entire protein. After culture, naïve-derived Th cells robustly recognized LAA in most donors, while bulk-derived CD4+ T cells had negligible activity, suggesting that pre-exiting memory Th cells have a competitive advantage over LAA-specific precursors. Addition of inflammatory cytokines (IL-1, 6, 7, 21, 23 and TGF-β) further enhanced reactivity. Naïve-derived donor LAA-specific Th cells (but not control CMV pp65-specific Th cells) specifically recognized HLA-matched sibling AML blasts. After adoptive transfer LAA-specific T cells significantly reduced leukemic burden in NSG mice bearing fully-HLA matched AML. Thus, it is feasible to generate naïve-derived multi-epitope specific anti-leukemia CD4+ T cells from normal donors. Removal of competing memory Th cells and culture in inflammatory cytokines unmasks strong LAA-specific reactivity. These Th cells demonstrate highly specific recognition of naturally processed LAA in HLA-matched leukemic blasts and can control AML in vivo, establishing a foundation for CD4+ T cell immunotherapy for hematological malignancies.
Naïve CD4+ T Cells, Recruited by CCL18 Secreted by Tumor-Associated Macrophages, Differentiate Within Breast Tumors into Tumor-Infiltrating Regulatory T Cells
Authors: Song Erwei , Su Shicheng , Liao Jianyou , Zhao Qiyi , Lieberman Judy , Liu Qiang
Published: January 2023

The origin of tumor-infiltrating Tregs, critical mediators of tumor immunosuppression, is unclear. Here we show that human breast tumor-infiltrating Tregs and naive CD4+ T cells have overlapping TCR repertoires, which barely overlap with circulating Treg sequences, suggesting that intratumoral Tregs develop from naive T cells in situ rather than from recruited Tregs. Naive CD4+ T cells, whose abundance in tumors is linked to poor prognosis, are recruited to human breast tumors when their PITPNM3 receptor recognizes the tumor-associated macrophage chemokine CCL18. In tumor-bearing humanized mice, blocking naive CD4+ T cell tumor recruitment by knocking down PITPNM3 reduces tumor-infiltrating Tregs and inhibits tumor progression. These findings suggest that breast tumor-infiltrating Tregs arise from chemotaxis of circulating naive CD4+ T cells that differentiate into Tregs in the tumor microenvironment. Inhibiting naïve CD4+ T cell recruitment into tumors by interfering with PITPNM3 recognition of CCL18 may be an attractive strategy for anticancer immunotherapy.
Loss of immune tolerance is controlled by ICOS in Sle1 mice
Authors: Mittereder Nanette , Kuta Ellen , Bhat Geetha , Dacosta Karma , Cheng Lily , Herbst Ronald , Carlesso Gianluca
Published: January 2023

ICOS, a member of the CD28 family, represents a key molecule that regulates adaptive responses to foreign Ags. ICOS is prominently expressed on T follicular helper (TFH) cells, a specialized CD4+ T cell subset that orchestrates B cell differentiation within the germinal centers and humoral response. However, the contribution of ICOS and TFH cells to autoAb profiles under pathological conditions has not been thoroughly investigated. We used the Sle1 lupus-prone mouse model to examine the role of ICOS in the expansion and function of pathogenic TFH cells. Genetic deletion of ICOS impacted the expansion of TFH cells in B6.Sle1 mice and inhibited the differentiation of B lymphocytes into plasma cells. The phenotypic changes observed in B6.Sle1-ICOS–KO mice were also associated with a significant reduction in class-switched IgG and anti-nucleosomal IgG-secreting B cells compared with B6.Sle1 animals. An upregulation in the IFN gene signature early in the life of B6.Sle1 mice modestly correlated with increased levels of autoAbs. The level of VCAM-1, a molecule that was shown to be elevated in patients with SLE and in lupus models, was also increased in an ICOS-dependent manner in Sle1 mice and correlated with autoAb levels. The elimination of ICOS-expressing CD4+ T cells in B6.Sle1 mice, using a glyco-engineered anti-ICOS–depleting Ab, resulted in a significant reduction in anti-nucleosomal autoantibodies. Our results indicate that ICOS regulates the ontogeny and homeostasis of B6.Sle1 TFH cells and influences the function of TFH cells during aberrant germinal center B cell responses. Therapies targeting the ICOS signaling pathway may offer new opportunities for the treatment of lupus and other autoimmune diseases.
CXCR3 blockade impedes the development of Sjögren’s syndrome-like xerostomia in non-obese diabetic mice
Authors: Zhou Jing , Yu Qing
Published: January 2023

The chemokine receptor CXCR3 plays an important role in T cell recruitment in various immune responses and autoimmune diseases. Expression of CXCR3 ligands, including CXCL9, CXCL10 and CXCL11, is elevated in the salivary glands of patients with Sjögren’s syndrome (SS). To elucidate whether interaction between CXCR3 and its ligands is required for the development of SS, we administrated an anti-CXCR3 blocking antibody to non-obese diabetic (NOD) mice, a well-defined model of SS, during the stage prior to disease onset. CXCR3 blockade significantly improved salivary secretion, indicating a remission of SS clinical manifestation. CXCR3 blockade did not affect the gross leukocyte infiltration of the submandibular glands (SMG) as assessed by H&E staining. However, flow cytometric analysis showed that anti-CXCR3 treatment markedly reduced the percentage of CXCR3+CD8 T cells and CD44+CD8 T cells, without affecting that of CXCR3+CD4 T cells and CD44+CD4 T cells in the SMG and submandibular lymph nodes, suggesting a selective effect of CXCR3 blockade on effector T cytotoxic 1 cells. Meanwhile, SMG expression of inflammatory factor TNF-α was markedly diminished by anti-CXCR3 treatment. Consistent with this, CXCR3 blockade significantly enhanced SMG expression of tight junction protein claudin-1 and water channel protein aquaporin-5, two factors that are crucially required for normal salivary secretion and can be down-regulated by TNF-α. Taken together, our findings demonstrated that the interaction between endogenous CXCR3 and its ligands plays a proinflammatory and pathogenic role in the development of SS-like xerostomia in NOD mouse model.
IL-17A inhibits expression of IL-17-lineage cytokines through a negative feedback loop involving IL-24 and controls autoimmune uveitis
Authors: Chong Wai Po , Raychaudhuri Kumarkrishna , Horai Reiko , Silver Phyllis , Jittayasothorn Yingyos , Chan Chi-Chao , Chen Jun , Caspi Rachel R
Published: January 2023

The Th17 response has been associated with autoimmune diseases in patients and in animal models. IL-17A is recognized as the Th17 signature cytokine and IL-17A-producing T cells are pathogenic effectors in models of autoimmunity, including experimental autoimmune uveitis (EAU). Paradoxically, however, injection of IL-17 ameliorates EAU (PMID: 19234216). Using a model of spontaneous uveitis in IRBP T cell receptor transgenic R161H mice, we investigated their susceptibility on the IL-17A−/− background. Surprisingly, IL-17A−/− R161H mice developed essentially undiminished uveitis and IL-17−/− R161H T cells, polarized to Th17 and infused into wild type recipients, induced similar disease to IL-17A sufficient R161H T cells. Interestingly, IL-17A−/− R161H T cells polarized under Th17 conditions produced elevated amounts of other Th17-related cytokines, i.e. IL-17F, GM-CSF and IL-22. Supplementing these cultures with recombinant IL-17A normalized the elevated production of these cytokines. RNAseq analysis revealed that IL-17A−/− T cells expressed lower levels of IL-24 compared to their IL-17A sufficient counterparts. Mechanistic studies indicated a negative feedback loop where IL-17A induces Th17 cells to produce IL-24, which subsequently suppresses production of Th17 lineage cytokines. Finally, injection of recombinant IL-24 ameliorated adoptive Th17-induced EAU, and conversely, silencing IL-24 expression in Th17 cells increased their pathogenicity and enhanced disease severity. Our data suggest that: (a) IL-17A exerts a negative feedback on uveitogenic Th17 cells via IL-24 production, and (b) IL-24 limits the expression of other Th17-related cytokines and controls their pathogenicity.
S1P1 Agonist SEW2871 Inhibits human Th17 cell generation and functions
Authors: Eken Ahmet , Okus Fatma Z , Yetkin Mehmet F , Cakir Mustafa , Ozdarendeli Aykut , Mirza Meral
Published: January 2023

S1P1 is a G-protein coupled receptor expressed by lymphocytes and is required for their egress out of secondary lymphoid organs. Fingolimod, an agonist of four S1P receptors including S1P1, is used in multiple sclerosis (MS) treatment in humans; however, less is known regarding the impact of S1P1 agonists on the egress-independent biology and functions of human Th17 and Treg cells. In this study, we explored the effects of S1P1 agonists SEW2871 and FTY720 on the ex vivo generation and functions of human Th17 and Treg cells. We report that a selective S1P1 agonist SEW2871 inhibited human Th17 development ex vivo, from naïve T cells, associated with reduced IL-17 and IL-22 production, demonstrated by ELISA and intracellular cytokine staining. On the other hand, Treg generation was augmented in SEW2871+ cultures. Additionally, SEW2871-exposed monocytes and dendritic cells produced less IL-23, a cytokine required for Th17 cell-expansion and pathogenicity. Th17 differentiation in the presence or absence of antigen presenting cells revealed that SEW2871 could inhibit Th17 generation either by blocking APC-derived IL-23 or directly affecting T cells. Our data provide novel information as to egress-independent effects of S1P1 agonists on Th17 cells.
Cellular misfolded proteins transported to the cell surface by aberrantly expressed MHC class II molecules are major autoantibody targets in autoimmune diseases
Authors: Arase Hisashi , Jin Hui , Tanimura Kenji , Suenaga Tadahiro , Arase Noriko , Lanier Lewis L
Published: January 2023

Cellular misfolded proteins are generally not transported outside the cells and thus may not be exposed to immune cells. This suggests that immune cells may not be tolerant to the cellular misfolded proteins. However, we recently found that such misfolded proteins in the endoplasmic reticulum are rescued from protein degradation and are transported to the cell surface by aberrantly expressed MHC class II molecules without being processed to peptides. Because structures of misfolded proteins associated with MHC class II molecules are different from those of normal proteins, their antigenicities are also different. On the other hand, it has been well known that MHC class II molecules are aberrantly expressed in many autoimmune diseased tissues. In this study, we extensively analyzed misfolded proteins transported to the cell surface by MHC class II molecules in autoimmune diseases and found that these misfolded proteins complexed with MHC class II molecules are specific targets for autoantibodies produced in autoimmune diseases such as rheumatoid arthritis and antiphospholipid syndrome. Furthermore, a strong correlation has been observed between autoantibody binding to misfolded protein/HLA-DR complex and the autoimmune disease susceptibility conferred by each HLA-DR allele, suggesting that misfolded protein/HLA-DR complexes are involved in the pathogenicity of autoimmune diseases. These findings imply that misfolded proteins rescued from protein degradation by aberrantly expressed MHC class II molecules function as a ‘neo-self’ antigens that might induce abnormal immune response in autoimmune diseases.
Visual function in retina-specific TCR-transgenic mice with spontaneous autoimmune uveitis
Authors: Jittayasothorn Yingyos , Horai Reiko , Caspi Rachel R
Published: January 2023

Autoimmune uveitis is a potentially blinding disease caused by circulating retina-specific T cells. To understand the mechanisms behind autoimmune uveitis, R161 transgenic mice expressing a TCR specific for IRBP, the retinal target protein for autoimmune uveitis in the mouse model, were generated. Three lines of R161 mice expressing the same TCR at different copy numbers have been established: R161H>R161M>R161L. The H and M lines developed spontaneous uveitis with 100% incidence by 2–3 months, whereas the L line developed disease only upon immune perturbation. The expression levels of the R161 TCR transgene correlated with frequency of IRBP-specific CD4 T cells in the peripheral repertoire, and with severity of disease. To study the association of visual function with pathology of uveitis, we examined visual function of R161 lines and compared with their parental strain B10.RIII (WT) by electroretinography (ERG), which measures electrical responses of retinal cells under dark- and light-adapted conditions. Uveitis was monitored and scored weekly by fundoscopy and ERG was performed in the next day following dark adaptation. Overall, as inflammatory scores increased, ERG responses decreased. R161L mice, which develop little or no disease, had visual function equivalent to WT mice. At 16 weeks old when disease in the H and M lines had reached its peak, R161H mice exhibited no and most of R161M mice exhibited poor visual function, whereas only minor reduction of ERG, which is normally observed with age, was detected in R161L and WT mice. The ERG data illustrate an association of visual function with uveitis scores among R161 lines, each of which may serve as a unique model of uveitis with different severity and visual loss.
Abacavir competes for HLA-B*57:01 restricted peptide presentation in HLA-transgenic mice.
Authors: Tilahun Mulualem E , Natarajan Kannan , Boyd Lisa F , Cardone Marco , Norcross Michael A , Margulies David H
Published: January 2023

A wide variety of genetically determined autoimmune phenomena and adverse drug reactions (ADR) are linked to specific HLA class I or class II genes. A particularly severe ADR is caused by the anti-HIV drug abacavir and is associated with HLA-B*57:01 expression. We have produced several transgenic mouse lines on the C57BL/6 background expressing HLA-B*57:01 as: 1) a full length HLA-B*57:01 protein; 2) a chimeric version in which the α3, transmembrane (TM) and cytoplasmic (CY) domains derived from H2-Dd; and 3) a single-chain molecule consisting of human β2-microglobulin linked to a chimeric version, HLA-B*57:01α1α2 and the H2-Dbα3, TM, and CY domains. Flow cytometry showed cell surface expression on all lymphoid cell populations tested. Expression of the transgenes on the H2-Kb−Db− background was significantly higher than in MHC-I sufficient mice. Immunization with an HLA-B*57:01-restricted peptide induced a CD8+ T-cell response, indicating the biological antigen presentation function of the transgenically expressed protein, and abacavir inhibited the peptide specific response. Furthermore, in vivo administration of abacavir followed by in vitro culture induced a CD8+ T-cell response and production of proinflammatory cytokines in the transgenics and not in controls. Thus, transgenic strains expressing HLA-B*57:01 present a specific HLA-B*57:01-restricted peptide, presentation is inhibited by abacavir, and abacavir alone can elicit CD8+ T cell responses. These observations support the view that HLA-B* 57:01 expression uniquely confers the abacavir sensitive phenotype. Such transgenic mouse strains provide useful models for exploring the mechanisms of HLA-B*57:01-linked adverse drug hypersensitivity reactions.
A novel flow cytometric assay for single-cell analysis of microRNA, messenger RNA and protein expression during cell differentiation and maturation.
Authors: Lai Chunfai , Stepniak Dariusz , Sias Leslie , Funatake Castle
Published: January 2023

We describe a novel assay that enables analysis of microRNA, mRNA, and protein expression in single cells by flow cytometry. This in situ hybridization-based method employs pairs of target-specific probes, branched DNA amplification technology, and fluorochrome-conjugated label probes, resulting in sensitive detection of different types of RNA targets including mRNA, microRNA, long non-coding RNA and viral RNA. To broaden our understanding of the applicability of this assay, we determined its limit of detection for mRNA and microRNA. Using in vitro bone marrow-derived macrophages as a model, we demonstrate how this assay can be used to study the process of cell differentiation and maturation. This novel assay enables the simultaneous detection of up to four RNA targets in the presence of fluorochrome-conjugated antibodies in single cells and is a powerful new tool that will enable new insights into cellular processes.
A method for determining antibody-dependent cellular phagocytosis
Authors: Kamen Lynn A , Kho Elviza , Ordonia Benjamin , Langsdorf Chris , Chung Shan
Published: January 2023

Antibody-based therapeutics are a powerful tool to treat diseases. While their mechanism of action (MOA) always involves binding to a specific target via the Fab region of the antibody, the induction of effector functions through the Fc region of the antibody is equally important for antibody therapeutics designed to deplete tumor cells. By binding of the Fc region to FcγRs on the surface of immune cells, antibody therapeutics exert effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), both of which induce target cell death and aid in the efficacy of the treatment. There are many in vitro assays designed to measure ADCC and CDC activity of therapeutics. However, another major Fc effector function is antibody-dependent cellular phagocytosis (ADCP). ADCP is the mechanism by which antibody-opsonized target cells activate the FcγRs on the surface of macrophages to induce phagocytosis, resulting in the internalization and degradation of the target cell through acidification of the phagosome. ADCP has been implicated as a major MOA of several biologics, but this activity is difficult to measure in an in vitro assay. Most assays measure the association between target cells and macrophages, however co-localization does not indicate phagocytosis. In this presentation, we describe the development of a robust method for measuring ADCP activity. By labeling target cells with a pH sensitive dye that only fluoresces in mature phagosomes, it is possible to quantitate the ADCP activity of different antibody therapeutics. This method was developed on parallel platforms, utilizing both flow cytometry as well as a novel method quantitating the ADCP activity on a cell imaging system.
Immunomagnetic separation technology with reversible labeling for direct isolation of leukocytes
Authors: Evaristo Cesar , Steinbrueck Philipp , Bissa Ursula , Yu Zhong , Brieden Jennifer , Dose Christian
Published: January 2023

Immunomagnetic enrichment of leukocytes plays an important role in both research and clinical applications. Accordingly, there is a continued need for improved and reliable cell separation methods. Current enrichment strategies with specific magnetic labeling of the target cells allow for efficient isolation but have the disadvantage of residual labeling on the cell surface after isolation. Therefore, the isolation of viable cells with a minimal potential of surface-initiated activation becomes increasingly more important.

To this end, we have combined magnetic cells separation, MACSTM, the proven state-of-the-art method for the isolation of viable cells from heterogeneous cell populations with a releasable-label technology developed at Miltenyi Biotec, which allows direct isolation of truly unmarked cells. This method provides an easy and fast solution for the highly specific separation of unlabeled leukocytes directly from PBMCs. In this poster we present our latest results from this novel technology applied to key markers in the immunology field.
A real-time digital bio-imaging system to quantify cellular cytotoxicity as an alternative to the standard chromium-51 release assay
Authors: Braud Veronique M , Fassy Julien , Tsalkitzi Kyriaki
Published: January 2023

Reliable measurement of cellular cytotoxicity is essential for the characterization of immune responses and for the monitoring of antibody treatment efficacy. Until now, the standard chromium 51Cr-release assay has remained the sole sensitive assay that measures cellular cytotoxicity. Alternative non-radioactive assays have been developed but they do not provide accurate measurement of target cell cytotoxicity. The cost and hazard of handling radioactivity are strong incentive to find alternative solutions to 51Cr. We took advantage of the recent development of cell-imaging multimode readers to develop a novel non-radioactive and real-time cytotoxic assay that demonstrates good reproducibility and sensitivity. The extent of target-cell cytotoxicity is monitored over time by imaging and quantifying live fluorescent target cells in 96-well plates using the cell-imaging multimode reader Cytation™ 5 from Biotek. We have developed classical NK cell assays in the presence or absence of blocking antibodies and antibody-dependent cell-mediated cytotoxicity (ADCC). We show that in these assays, cell killing occurs within the first two hours with a half maximum killing reached after 30 minutes. This technology has numerous applications such as NK and T cell cytotoxicity assays and can be extended to cell survival and apoptosis measurement assays.