bluuebunny/crossref_metadata_2025 huggingface dataset:
Prenatally Educated Friendly NK Cells regulate Allo-specific T cell Responses through expansion of Foxp3+CD4+Treg Cells and suppression of Type 1 and 17 CD8+T cell responses
Authors: Kang Hee Kap , Papastefan Steven T , Turner Lucas , Redden Catherine , Langereis Morgan , Alhajjat Amir , Shaaban Aimen
Published: August 2023

Prenatal alloantigen exposure during embryonic development permanently tolerizes the early gestational fetus and provides a tremendous advantage over transplantation after birth. However, the influence of prenatally educated NK cells on alloantigen-specific T cell immune responses is not well elucidated. In previous experiments, we found that prenatally-educated friendly NK (fNK) cells exert suppressive effects on allo-specific T cell responses in vitro. We hypothesized that prenatally-educated fNK cells exert regulatory effects on allo-specific T cells in vivo.

To challenge this hypothesis, we examined the regulatory potential of prenatally educated fNK cells through gain and loss of function experiments in stable prenatal Balb/c --> B6.Thy1.2+ chimeras (engrafter mice). We depleted both (Ly49AFG+) fNK cells and Thy1.2+T cells or Thy1.2+T cells alone in host engrafter mice and then adoptively transferred naïve responder Thy1.1+ T cells along with donor-specific allogeneic or syngeneic target cells into the engrafter hosts. We found that the fNK cell-replete animals exhibited a higher frequency of foxp3+Tregs and a lower frequency of allospecific T-bet+ and RORγt+ CD8 T cells compared to the fNK cell-depleted controls. Furthermore, the neutralization of TGF-β1 decreased the fNK cell-induced expansion of Treg cells in vitro.

From these studies, we conclude that prenatally educated fNK cells exert regulatory effects on allo-specific T-cell responses through the TGF-β1 mediated expansion of foxp3+Treg cells. Prenatal antigen experience drives the emergence of antigen-specific suppressors that regulate T cell responses.

National Institutes of Health Grant R01HL103745 and Lurie Children’s Hospital Research Foundation (to A.F.S.)
Zinc-c-di-AMP nanoparticles target and suppress tumors via endothelial STING activation and tumor associated macrophage reinvigoration
Authors: Yang Kaiting , Han Wenbo , Jiang Xiaomin , Piffko Andras , Bugno Jason , Han Chuanhui , Li Sirui , Liang Hua , Xu Ziwan , Zheng Wenxin , Wang Liangliang , Wang Jiaai , Huang Xiaona , Ting Jenny P , Fu Yang-Xin , Lin Wenbin , Weichselbaum Ralph R
Published: August 2023

Mounting evidence indicates that the stimulator of interferon genes (STING) pathway is critical for the effectiveness of standard cancer treatments. However, systemic delivery of STING agonists remains a challenge due to poor tumor-targeting and unwanted toxicities. Here we describe a robust tumor-targeted STING agonist, ZnCDA, formed by the encapsulation of bacterial-derived cyclic dimeric adenosine monophosphate (CDA) in nanoscale coordination polymers. Intravenously injected ZnCDA prolonged CDA circulation and efficiently targeted tumors, mediating robust antitumor effects in a diverse set of preclinical cancer models. Moreover, endothelial STING activation promotes the accumulation of ZnCDA in the tumor tissues by amplifying the enhanced permeability and retention (EPR) effect. In this study, we discover that type I IFN is not required for the antitumor effects of STING agonism by ZnCDA, indicating that differentially formulated STING activators may suppress tumor growth in different ways. Using metal- or dye-labelled nanoparticles, we identified a group of tumor-associated macrophages (TAMs) as the cellular target of ZnCDA in the tumor immune microenvironment. RNA-seq analysis indicated the modulation of TAMs for antigen processing and presentation via STING activation. We further showed that TAMs with ZnCDA uptake promoted an antitumor T cell response. Lastly, we utilized two immunologically “cold” tumor models, a fragment Panc02-SIY pancreatic cancer model and an intracranial GL261 glioma model, both of which represent intractable fatal human cancers, to provide a proof-of-concept strategy for combining ZnCDA with immune checkpoint blockade or radiotherapy in aggressive, difficult-to-treat tumors.

a grant from the Ludwig Foundation and an NIH grant (R01CA262508) to R.R.W., NIH grants (R01CA223184 and R01CA216436) to W.L. and NIH grants (R35CA232109 and R01AI141333) to J.P.Y.T. A.P. is the recipient of a Walter Benjamin scholarship provided by the German Research Foundation (Deutsche Forschungsgemeinschaft). J.B. was supported by a Clinical Therapeutics Training Grant (T32GM007019).
High-intensity focused ultrasound ablation increases lymphocyte infiltration in mouse prostate tumor
Authors: Su Shengchen , Wang Yanping , Lo Eric M , Tamukong Patrick , Kim Hyung L
Published: August 2023

Introduction and Objective

High-Intensity Focused Ultrasound (HIFU) is an FDA-approved procedure for the treatment of localized prostate cancer. HIFU destroys tumor tissue by noninvasively delivering ultrasonic energy, which rapidly heats the targeted tissue. Prostate cancer has few tumor-infiltrating lymphocytes, which are required for antitumor immunity. The objective of this study was to determine if HIFU activates antitumor immunity and increases lymphocyte infiltration into the tumor.

Methods

RM1 tumors were grown on the flanks of syngeneic C57BL/6J mice and one tumor was treated with HIFU. Contralateral tumor growth was monitored, and tumors were collected at the end of the study to assess immune cell infiltration. Tumor infiltrating lymphocytes were assess by flow cytometry.

Results

When one tumor was treated with HIFU, the growth of the contralateral (untreated) tumor decreased when the contralateral tumor was the same tumor type but not when it was a different tumor type. HIFU increased CD4+ and CD8+ lymphocyte infiltration into the contralateral (untreated) tumor. Depletion studies confirmed that the antitumor immune effect of HIFU is CD4+ and CD8+ lymphocyte dependent.

Conclusions

In preclinical models, HIFU generates an antitumor immune response capable of suppressing the growth of mouse prostate tumors. The antitumor immune effect of HIFU is CD4+ and CD8+ lymphocyte dependent. Future studies should investigate mouse models of metastatic prostate cancer, where cytoreductive prostate tumor ablation can be combined with systemic immunotherapies to generate robust antitumor immunity.

Homer and Gloria Harvey Family Chair Fund in urologic oncology in honor of Stuart Friedman, M.D.
Depletion of plasmacytoid dendritic cells and HIF-1α inhibition enhance immunotherapy against hepatocellular carcinoma
Authors: Geng Degui , Wang Yin , Liu Minyue , Ahodantin James , Li Guangming , Su Lishan
Published: August 2023

Hepatocellular Carcinoma (HCC) is one of the deadliest forms of cancer. Recent years have seen significant advances for HCC treatment, but up to 70% of patients remain at high risk for recurrence necessitating the urgent need for more effective therapies. Plasmacytoid dendritic cells (pDCs) heavily infiltrate liver tumor tissues, contribute to an immunosuppressive tumor microenvironment (TEM), and promote HCC growth. Furthermore, hypoxia often occurs in HCC tumor tissues and plays the central role of an immunosuppressive TME. A hypoxic TME enhances pDC recruitment into tumor tissues via the upregulation of hypoxia-inducible factor 1α (HIF-1α). Here we show that the distribution of pDCs does not co-localized with hypoxic areas in HCC. In the immunocompetent HCC mouse model, depletion of pDCs using the antibody or HIF-1α inhibitor echinomycin significantly suppressed tumor growth. The combination of pDC depletion and HIF-1α inhibition suppressed HCC growth more effectively. Both treatments reshaped the TME with changes of the cytokine profile and increased tumor-infiltrating CD8 T cells. Reduced basic helix-loop-helix transcription factor E2-2 expression by treatments abrogated protumor functions of pDCs in HCC resulting in reactivating tumor-reactive CD8 T cells. This study demonstrates that targeting both pDCs and HIF-1α may serve as more effective immunotherapy for HCC.
Avian paramyxovirus 4 has potent antitumor activity that surpasses the oncolytic effect of NDV in colon carcinoma and melanoma in vivo models
Authors: Bykov Yonina , Javaheri Aryana , Mena Ignacio , Garcia-Sastre Adolfo , Cuadrado-Castano Sara
Published: August 2023

Avulaviruses make up some of the most widespread avian viral pathogens. Within the genus, APMV-1, also known as Newcastle disease virus (NDV), is the only avulavirus that has been extensively characterized and explored as an oncolytic agent. While an array of clinical and preclinical studies have shown NDV to hold much promise, the oncolytic potential of the other avulaviruses remains largely unexplored. In this study, we evaluated the in vivo anti-tumor potential of prototype strains of APMV serotypes -2, -3, -4, -6, -7, -8 and -9 in syngeneic B16–F10 melanoma and CT26.WT colon carcinoma models. All the selected viruses were able to delay tumor growth, however, treatment with APMV-4 Duck/Hong Kong/D3/1975 imparted the greatest survival benefit as well as a number of complete remissions that were protected against re-challenge in both tumor models. Infection of cancer cell lines with APMV-4 in vitro resulted in potent activation of the IFN response, and in vivo interrogation of the tumor microenvironment revealed high levels of infiltration by T cells as well as various members of the myeloid compartment. Importantly, the development of a reverse genetics approach to rescue recombinant APMV-4 viruses allowed us to successfully express GFP in the virus backbone, highlighting APMV-4 as a versatile tool that can, in the future, include therapeutic transgenes that will optimize its potential benefit in the clinic.

supported in part by a Public Health Service Institutional Research Training Award AI07647
Combination epigenetic therapy activates immune cells in a murine model of ovarian cancer
Authors: Cox Olivia L , Gomez Stephanie , Grundy Erin E , Chiappinelli Katherine B
Published: August 2023

Ovarian cancer (OC) is the fifth leading cause of cancer death in women in the United States. Despite novel therapies and significant advances in our understanding of the disease, the five-year survival rate for OC has not changed in decades. The immunosuppressive microenvironment of OC leads to a low response rate to immunotherapies. We have previously shown that in a murine model of OC epigenetic therapies including DNA methyltransferase inhibitors (DNMTis) and histone deacetylase inhibitors (HDACis) activate type I interferon signaling in tumor cells to drive immune infiltration and sensitization to immunotherapy. This indicates a role for immune control of the tumor, but we did not directly investigate the effects of these epigenetic therapies on the immune cells. We hypothesized that T cells could be activated by epigenetic therapy in this model.

We isolated T cells from the spleens of healthy C57/Bl6 mice and treated the cells with a DNMTi, 5-azacytidine (Aza) at 500 nmol/L, and an HDACi, ITF-2357 (ITF) at 100 nmol/L, singly and in combination. Cell pellets were isolated, RNA extracted, and analyzed via qRT-PCR. Cell supernatants were analyzed for cytokine and chemokine production using a Legendplex assay. The treated bulk T cells, CD8+ T cells, and CD4+ T cells showed anti-tumor cytokines, including CCL5, TNF-α and IFN-γ, were upregulated while the pro-tumor cytokine IL-10 was downregulated. These changes were evident at the transcript and secreted cytokine level, pointing to transcriptional upregulation of the target genes. This study demonstrates direct activation of T cells by DNMTis and HDACis and provides mechanistic insight into the role of epigenetic therapies in the murine model of OC.
Sensitization of Tumor Cells to IFNy-Mediated Cell Death
Authors: Sun Nicholas , Krogman Erica , Chawla Yogesh , Ting Adrian
Published: August 2023

There is increasing evidence that immunotherapies make tumor cells more vulnerable to the host’s own immune system that can lead to long-term and durable remission. To date, only a small percentage of cancer patients have benefited from this approach and advances in this field will likely come from a greater understanding of the interaction between immune cells and their tumor targets. T cells are critical to a successful anti-tumor response due to their ability to directly lyse tumor cells by releasing cytolytic granules, and to produce inflammatory cytokines, such as interferon gamma (IFNγ). IFNγ has several functions in an anti-tumor response. On the immune cell side, IFNγ is a major driver of Th1 differentiation that leads to the activation of T cells and NK cells. On the tumor side, IFNγ can enhance MHC class I presentation but also inhibitory receptors such as PD-1. However, it is not as clear whether IFNγ is directly cytotoxic for tumor cells. If that is the case, it is also likely that due to selection pressure exerted by T cells, tumor cells may have acquired mechanisms to protect themselves against this possibility. Thus, I hypothesized that tumor cells are normally resistant to killing by IFNγ but can be sensitized if the death protection mechanisms are disabled. In preliminary studies using the B16F1 murine melanoma model, we found that while wild-type (WT) melanoma cells were completely resistant, tumor cells with knockouts of two kinases (DKO) became highly sensitized to IFNγ-mediated apoptotic death. Furthermore, the DKO tumors were also more easily killed by T cells in an IFNγ-dependent manner. These findings will provide insight into potential targets for therapeutic intervention to enhance immune-mediated destruction of tumors.
An in vitro T cell Dysfunction/Exhaustion Assay System that is partially reversible by pembrolizumab
Authors: Daftarian Pirouz M , King Jeffrey , Goverse Gera , Kleiman Eden
Published: August 2023

The persistence of antigen and chronic stimulation of T cells results in T cells’ functional decay, referred to as T cell dysfunction or exhaustion (TEX). TEX is an essential part of the checks and balances of the immune system chronic T cell stimulation. Assays that model T cell dysfunction/exhaustion with functional readouts are urgently needed for identification and characterization of active drug candidates.

Here, to recapitulate persistent of antigen as it happens in tumor microenvironment, we have optimized an in vitro stimulation set-up using PBMCs from various donors that models key features of T cell exhaustion/dysfunction including main markers and functional features of T cell exhaustion. Various stimulation conditions were assessed, with stimulation lasting up to four rounds, and optimal conditions were selected for anti-CD3/anti-CD28 stimulations of PBMCs that resulted in a) increased expression of the co-inhibitory molecules associated with exhausted T cells, PD-1, TIM3, LAG3, CD38, and CD39, and b) impaired T cell functions as demonstrated by reduced expression of IFN-γ, TNF-α, and reduced cytotoxic killing in an allogeneic setting, using both a 2D cancer cell line and a 3D tumoroid model. This in vitro T cell dysfunction/exhaustion platform is a candidate to test drug candidates for their ability to delay or partially reverse T cell exhaustion.

Crown Bioscience internal R&D
A 2-laser 13-color cFluor®B Cell Monitoring panel for analyzing human B cell subsets
Authors: Miller Heather , Tran Kevin , Edinger Mark , Chen Jingyi , Deng Jun
Published: August 2023

Flow cytometry is valuable for identifying and monitoring B cell subsets in patients with humoral immune disorders and patients treated with immunotherapies targeting B cells. We have designed a 13-color cFluor®B Cell Monitoring panel that identifies plasmablasts/plasma cells (CD19+ CD20− CD27+ CD38++), naïve B cells (CD27− IgD+), IgG class switched memory (CD27+ IgG+) and unswitched memory (CD27+ IgD+ and/or IgM+). Additionally, markers for CD4+ and CD8+ T cells are included for a general status of lymphocytes along with granulocyte (CD15) and monocyte (CD14) markers to allow for cleaner lymphocyte gating.

The panel was tested on healthy PBMCs and peripheral blood samples collected in EDTA, Cyto-Chex BCT, or heparin tubes. The cell populations of interest were well identified in the PBMCs and blood samples collected in the different types of blood tubes. The resolution of each marker in the panel was comparable to the single-color staining. Evaluation of assay reproducibility showed that the coefficient of variance (CV) for the B cell subsets of interest were <15% in triplicate runs of 10 samples. Also, blood sample stability and post staining stability were evaluated over time.

In conclusion, Cytek’s 13-color cFluor®B Cell Monitoring panel was shown to be effective in identifying and enumerating B cell subsets. This may be a useful tool for monitoring B cell subsets of patients with humoral immune disorders or patients treated with immunotherapies targeting B cells in translational research.
Impact of type I IFN signaling on B cell pathogenicity in a mouse model of early Sjögren’s disease
Authors: Allred Merri-Grace , Lieberman Scott M
Published: August 2023

Sjögren’s disease is a chronic autoimmune disease characterized by focal inflammation of the lacrimal and salivary glands. NOD mice spontaneously develop lacrimal gland inflammation by 5–6 weeks. As disease progresses, B cells accumulate in the glands and produce antibodies resulting in glandular dysfunction. Disruption of the type I interferon (IFN) signaling pathway protects NOD mice from developing lacrimal gland disease. While the impact of B cells later in disease has been well-characterized, the role of B cells in early disease development has not been defined. Here, we performed gene expression studies, adoptive transfers, and spectral flow cytometry to define the phenotype of B cell subsets and the potential role of type I IFN signaling in lacrimal gland autoimmunity.

Gene expression studies demonstrated that genes associated with B cell receptor signaling, lymphocyte trafficking and antigen presentation were upregulated at 10 weeks compared to 6 weeks. Flow cytometry of the lacrimal glands showed that most early B cells express IFNAR1. Adoptive transfer studies demonstrated decreased disease when donor cells lacked IFNAR1 expression compared to WT donor cells, but this difference was abrogated when magnetically purified T cells were transferred suggesting a type I IFN dependent pathogenic role on a non-T cell population. In vitro studies show that B cell phenotype can be shaped by IFNα in a dose dependent manner.

Type l IFN signaling drives the development of lacrimal gland autoimmunity in NOD mice. Type l IFN signaling shapes B cell phenotype and function in early lacrimal gland disease. Ongoing in vivo studies will interrogate the impact of IFNAR1 expression on B cell trafficking to the lacrimal glands.

Supported by grants from NIH (R01 EY027731)
Impaired expansion and selection of geminal center B cells in lupus drives autoantibody-producing cells
Authors: Sanchez Gina Marie , Antao Olivia , Mayer Daniel , Hirsch Eden , Lemenze Alexander , Weinstein Jason
Published: August 2023

Lupus is a systemic autoimmune disease characterized by the presence of autoantibodies that form immune complexes in tissues leading to the recruitment of immune cells and subsequent inflammation and organ damage. The origin of autoantibody-secreting B cells is not clear, but the germinal center (GC) is of particular interest as the B cells here undergo somatic hypermutation, which may result in the generation of autoreactive memory or long-lived plasma cells. Within the GC, B cells cycle between two major zones: the dark zone (DZ) where mutation and proliferation occur, and the light zone (LZ) where affinity-based selection occurs. We found in lupus-prone mice that as autoantibodies increased, there was a reduction in proliferation measured by EdU incorporation in conjunction with dysregulation of the GC zones as B cells here became skewed towards a LZ phenotype (CXCR4lo CD86+). As MYC expression in LZ B cells correlates with the strength of selection, we examined MYC levels by flow cytometry and Amnis and found it and its nuclearization decreased as disease progressed. Single cell BCR sequencing of GC B cells throughout the course of disease revealed a loss in BCR repertoire diversity and mutations. Furthermore, we found that despite the accumulation of LZ B cells, there was an increase in the generation of plasma cells and memory-like CD11c+ Tbet+ B cells, suggesting aberrant selection and differentiation into these potentially autoantibody-secreting cells. Thus, our findings suggest that as lupus disease progresses, the ability of GC B cells in the LZ to undergo proper selection is impaired, resulting the permissive selection of autoreactive B cells and differentiation into autoantibody-secreting cells.

Supported by grants from the NIH/NIAMS (R01 AR073912, R01 AR073912 - 03W1)
Rheumatoid arthritis associated with a cytotoxic cell leukemia is characterized by enhanced reactivity to citrullinated antigens
Authors: Ananth Kusuma , Gomez-Banuelos Eduardo , Trejo-Zambrano Isabel , Kirschmann Jessica Marie , Moosic Katharine B , Wang Hong , Feith David J , Robinson William H , Andrade Felipe , Loughran Thomas P , Darrah Erika
Published: August 2023

T cell large granular lymphocyte leukemia (T-LGLL) is characterized by clonal expansion of cytotoxic T cells and neutropenia. Compared to 1% of the general population, about 25% of T-LGLL patients develop rheumatoid arthritis (RA), an autoimmune disease characterized by anti-citrullinated protein antibodies (ACPAs). To investigate the autoimmune response in patients with both T-LGLL and RA (T-LGLL/RA), we analyzed their ACPA positivity and specificity using anti-CCP ELISA (n=30 for T-LGLL/RA and T-LGLL; n=150 for RA) and multiplex array (n=26 per group), respectively. We found that anti-CCP positivity was enriched in patients with T-LGLL/RA (90%) compared to those with RA alone (75%; p=0.09), or T-LGLL alone (33%; p<0.0001). This was supported by multiplex serological profiling of reactivity to 51 individual RA autoantigens, which demonstrated that T-LGLL/RA patients are more responsive to citrullinated (cit)-antigens. T-LGLL/RA patients were overrepresented (61%) in a cluster of highly reactive individuals compared to patients with RA (39%) or T-LGLL alone (0%), and recognized a higher number of cit-antigens than patients with either disease alone: 11.7 vs. 5.8 for RA alone (p=0.2135) and 2.4 for T-LGLL alone (p<0.0001). Among specific antigens, recognition of a cit-clusterin and a cit-fibrinogen alpha peptide were higher in patients with T-LGLL/RA than RA alone (p=0.0253 and 0.0507, respectively). Our results show that T-LGLL/RA patients have more robust anti-CCP responses and recognize a wider diversity of citrullinated RA autoantigens than patients with RA or T-LGLL alone. Investigating immune responses in T-LGLL/ RA, which may consist of an extreme form of RA, may provide insight into RA pathogenesis.

This research was funded by NIH/NIAMS R01-AR079404.
Macrophage extracellular traps require peptidylarginine deiminase 2 and 4 and are a source of citrullinated antigens bound by rheumatoid arthritis autoantibodies
Authors: Bashar S Janna , Holmes Caitlyn , Shelef Miriam
Published: August 2023

Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis, but the sources of citrullinated antigens remain incompletely defined. Neutrophil extracellular traps (NETs) appear to be a source of ACPA targets. However, while the citrullinating enzyme peptidylarginine deiminase (PAD)4 is required for producing citrullinated NETs, it is not required for citrullination in murine arthritis. Conversely, PAD2 has a limited role in producing NETs but is a major contributor to citrullinated proteins. Thus, additional sources of citrullinated antigens likely exist. We investigated if macrophage extracellular traps (METs) could be a source of citrullinated proteins in a PAD2 or PAD4 dependent manner. Thioglycolate induced macrophages from wild-type, PAD2−/− and PAD4−/− mice were activated with a variety of stimulants, then fixed and stained with DAPI and either anti-citrullinated histone H4 (H4C3) antibody or sera from ACPA+ or ACPA− rheumatoid arthritis subjects. METs were visualized by immunofluorescence, confirmed to be extracellular using DNase, counted in a blinded manner, and compared by paired-T test. We found that ionomycin and monosodium urate (MSU) crystals induce citrullinated METs (H4C3 stained). In the absence of PAD2 or PAD4, citrullinated METs were reduced in response to ionomycin and MSU when compared to wildtype, with a complete absence of citrullinated METs in PAD4−/− macrophages. Also, IgG from ACPA+ sera, but not ACPA− sera, bound to METs. Finally, in the absence of PAD2 or PAD4, ACPA bound METs were reduced in response to ionomycin and MSU. Thus, METs appear to contribute to the generation of citrullinated antigens targeted by ACPAs in rheumatoid arthritis in a PAD2− and PAD4-dependent manner.

Funding for this work was provided by the U.S. Army MedicalResearch Acquisition Activity through the Peer Reviewed MedicalResearch Program (W81XWH-18-1-0717) to M.A.S.
Dissecting the functional role of SENP2 in B cells and systemic lupus erythematosus
Authors: Huang Chien-Hsin , Lin I-Ying , Lin Kuo-I
Published: August 2023

SUMOylation is a post-translational modification that can dynamically regulate protein activity. SUMOylation involves covalent binding to a lysine residue of the target proteins, and the SUMO-specific peptidase (SENP) catalyzes deconjugation. In our laboratory, we analyzed the changes in the expression of genes involved in SUMOylation and found a significant reduction of SENP2 in peripheral navie B cells isolated from patients with SLE as compared to that from healthy donors. We thus stimulated peripheral human B cells and transfected them with siRNAs against SENP2. Our results showed a significant increase in IgG antibodies after knocking down SENP2. We also stimulated naive splenic mouse B cells with T cell-independent (TI) or T cell-dependent (TD) stimulation to examine the expression level of SENP2 after B cell activation. The results showed downregulation of SENP2 protein after TD stimulation, but not after TI stimulation. Taken together, because the increase in IgG antibodies is often due to an enhanced germinal center (GC) response, we next investigated the role of SENP2 in B-cell differentiation and SLE pathogenesis. Hence, we developed a mouse model in which Senp2 is deleted in GC B cells. However, after immunization with TD stimulation, we found a reduction in the frequency of GC-B cells, plasma cells, IgG1-expressing cells, and the amount of antibody production in GC-specific Senp2 knockout mice, which can be recapitulated by using an in vitro induced germinal center culture (iGB) system. We thus hypothesized that the role of SENP2 for limiting IgG production may act in early-phase before GC B cells. The exact timing and mode of action of SENP2’s activity to restrain the production of IgG will be investigated.
CXCL4, a chemokine upregulated in systemic sclerosis patients, abrogates TLR9 signaling and central tolerance in B cells
Authors: Ah Kioon Marie Dominique , Cakan Elif , Garcia-Carmona Yolanda , Cunningham-Rundles Charlotte , Meffre Eric , Barrat Franck J
Published: August 2023

Background

Systemic sclerosis (SSc) is an autoimmune connective tissue disease, characterized by defective central B cell tolerance. Deficiency of MYD88, which mediates the function of TLRs, results in a failure to silence autoreactive B cells. CXCL4, a chemokine found elevated in the sera of SSc patients, potentiates IFN-α secretion from TLR9-induced plasmacytoid dendritic cells. The objective is to investigate whether CXCL4 regulates TLR9 signaling and central tolerance in B cells.

Methods

B cells from SSc patients or healthy donors were cultured with TLR9 ligand (TLR9-L) alone or with CXCL4. Cytokines gene expression and secretion were analyzed. Delivery of TLR9-L was analyzed by Amnis and confocal microscopy. NOD-scid-common gamma chain (γc) knockout immunodeficient mice were engrafted with CD34+ human fetal hematopoietic stem cells transduced with lentivirus expressing human CXCL4. New emigrant/transitional B cells were sorted. Immunoglobulins chains of these cells were cloned and transfected in fibroblasts. Polyreactivity of the antibodies released by the fibroblasts were assessed.

Results

Defective TLR9 signaling was observed in B cells of SSc patients. CXCL4 abrogated TLR9 response in human B cells. TLR9-L when given to B cells alone, was localized in the lysosomes and LAMP-2+ compartments while CXCL4 prevented the delivery of TLR9-L to these vesicles. CXCL4 in vivo expression led to defective TLR9 responses and increased number of polyreactive B cells.

Conclusions

Our data provide evidence for a novel mechanism by which CXCL4 impairs central B cell tolerance by altering the intracellular trafficking of TLR9 ligands, hence inhibiting TLR9 response which is required for the removal of developing autoreactive B cells.
17β-estradiol and B-cell intrinsic type I interferon amplify toll-like receptor 7 signaling loop in B cells of female lupus prone BXD2 mice
Authors: Sullivan Kathryn , Wang Yong , Chatham Winn W , Mountz John D , Hsu Hui-Chen
Published: August 2023

While systemic lupus erythematosus (SLE) disproportionally affects women versus men, elevation in 17β-estradiol (E2) alone is not sufficient to promote the development of autoantibody producing B cells. The objective of the present study is to determine if B-cell intrinsic mechanisms contribute to the increased TLR7 response to E2 stimulation. We identified that there were elevated circulating levels of E2 in young African American (AA) SLE patients (24–41 yr-old), compared to older AA (42–56 yr-old) and European American SLE patients (24–66 yr-old). Circulating E2 levels positively correlated with the levels of anti-Smith (Sm) and the expression of interferon-beta (IFNβ) in naïve B cells of SLE patients (n=39). Mouse studies were used to determine if E2 stimulates the expression of IFNβ in B cells and if sex plays a role to influence B cell responses to E2. Serum levels of Sm/RNP and RNP autoantibodies positively correlated with the levels of E2 in female lupus prone BXD2 mice post-puberty (12-wk-old) but not pre-puberty (4–6-wk-old). At the post-puberty stage (>12 wk-old), there were significantly elevated levels of anti-DNA and anti-Sm in female BXD2 mice, compared to male mice. This was associated with an increased TLR7-induced expression of IFNβ and CD69 in transitional stage 1 (T1: CD23− IgM+ CD93+) B cells in female BXD2 mice, compared to male mice. Interestingly, E2 stimulation promoted intracellular levels of IFNβ and TLR7 in T1 B cells from female but not male BXD2 mice. Together, our results suggest that elevation of E2 in combination with an increased B-cell susceptibility to E2 induction of IFNβ may play a role in the increased B cell responses to TLR7 stimulation in individuals predisposed to the development of SLE.

This study was supported by grants from VA Merit Review grant (I01BX004049), NIH grants R01-AI-071110, R01 AI134023, and Lupus Research Alliance Distinguished Innovator Award to J.D.M, the LRA Target Identification in Lupus Award to H-C.H., and the P30-AR-048311
OC-STAMP-reactive IgG auto-antibody may upregulate osteoclastogenesis in association with elevated anti-P. gingivalis antibody
Authors: Nakamura Shin , Shindo Satoru , Leon Elizabeth , Kumagai Tomoki , Pastore Maria Rita , Ouhara Kazuhisa , Kawai Toshihisa
Published: August 2023

Objectives

Periodontal disease (PD) is a polymicrobial inflammatory lesion in which pathogenic bone loss is caused by osteoclasts (OC) differentiated from MCSF/RANKL-primed monocytes. Elevated IgG antibody to a keystone opportunistic pathogen, Porphyromonas gingivalis (Pg), is a hallmark humoral response in PD that is correlated with pathogenic bone loss. Whereas, a cell membrane protein, osteoclast stimulatory transmembrane protein (OC-STAMP), is engaged in cell-fusion event required for OC-genesis. This study examined the humoral immune responses to OC-STAMP expressed by human monocytes in relation to anti-Pg antibody.

Methods

Blood serum (N=10) collected from systemically healthy human subjects (Precision for Medicine) were measured for IgG antibody titer to Pg or OC-STAMP peptides. Human peripheral blood mononuclear cells were stimulated with MCSF/RANKL with or without anti-OC-STAMP rabbit polyclonal antibody or control rabbit IgG. Affinity purified human anti-OC-STAMP antibody was also tested for its activity to alter the OC-genesis. OC-genesis induced in vitro was evaluated by TRAP staining and pit formation assay.

Results

IgG antibody titer to Pg positively correlated with that to human OC-STAMP peptides (r=0.579, p <0.05). Anti-OC-STAMP auto-antibody purified from human serum with high IgG antibody titer to Pg, as well as anti-OC-STAMP rabbit antibody significantly promoted in vitro OC-genesis (p <0.05).

Conclusions

The results indicated that Pg infection may be associated with development of anti-OC-STAMP auto-antibody which can promote OC-genesis, suggesting a novel immunological intervention in the pathogenesis of PD.

Supported by grants from NIH NIDCR grants, DE-027851, DE-028715 and DE-331851
Development of a seven-color immunofluorescent panel to identify a rare B cell population (IgM+ IgDlo) and its effect on T regulatory cell (Treg) proliferation
Authors: Beltrame Angela , Dittel Bonnie
Published: August 2023

Multiple sclerosis (MS) is caused by a T cell orchestrated immune response to myelin sheath antigens of the central nervous system. Autoimmune responses can be suppressed by CD4+ Foxp3+ T regulatory cells (Treg); therefore, a therapeutic strategy is to increase endogenous Treg numbers. Our studies using the mouse model of MS, experimental autoimmune encephalomyelitis, have identified a unique subset of IgM+ IgDlow/− B cells (BDL) that induce Treg proliferation in a glucocorticoid-Induced TNFR-related (GITR:GITRL)-dependent manner. However, where BDLlocalize with Treg in the spleen is unknown and imaging is challenging due to rarity of both cell populations. Because Treg remain within the T cell zone, we hypothesize BDL-Treg interactions take place within the T-B boundary. To visualize and localize BDL-Treg-interactions within the spleen, a seven-color microscopy panel was developed to identify splenic architecture using the Nikon Inverted Eclipse Ti2 microscope and SPECTRA Light Engine filter cube. Fluorescent conjugated antibodies to identify the marginal zone (MAdCAM-1), BDL(IgM, IgD), Treg (CD4, Foxp3), proliferation (Ki-67), and chemokine receptors (used for lymphocyte localization in the spleen) were selected. To select fluorescent antibodies with low background and spectral overlap thereby ensuring detection of real signal, cytospin slides of sorted CD19+ B cells or CD4+ T cells and single-stained controls were utilized. This method can be applied for a variety of imaging experiments and will be beneficial for other applications. By completing this project, we hope to better understand BDL-Treg interactions to develop a BDL-based therapeutic to increase Treg numbers and reduce autoimmunity.

MCW Center for Immunology GRIT Award
B cell apoptosis and inflammation conspire to promote lupus in mice
Authors: Khan Wasif N , Bazile Cassandra , Clark Emily S , Shaikh Kashif , López-Rodríguez Darlah M , LaPorte Kathryn M , Wright jacqueline A , Carlesso Gianluca , Riggs Jeff , Altman Norman , Greidinger Eric L , Malek Thomas R , Harhaj Edward W
Published: August 2023

The pathogenesis of Systemic lupus erythematosus (SLE) is driven by autoreactive B and T cells and autoinflammation. Impaired apoptosis in T and B cells has been shown to result in autoimmune disease, and we recently demonstrated that reducing apoptosis in B cells alone, by B cell-specific deletion of Bim (B.Bimf/f) could lead to SLE with prominent features of Sjogren’s Syndrome in C57BL/6 mice. Consistently, B cell targeting therapies are successful in reducing SLE pathogenesis in mouse models, however, their clinical success requires a better understanding of the contributions of B cells and the mechanisms that drive inflammation to autoimmunity. To address the role of inflammation in the development and progression of autoimmunity we generated a novel mouse model that lacks ZFAND6 (Zfand6−/−) and crossed with the B.Bimf/f mice. ZFAND6 is an A-20 like ZF domain-containing protein with a potential role in the negative regulation of NF-kB activation, however, its physiological function is unknown. The compound mutant mice displayed accelerated and exacerbated overlapping lupus and Sjogren’s autoimmune symptoms with a higher incidence of splenomegaly and kidney pathology than either single mutant alone including the early appearance of anti-SSA, anti-SSB, and anti-RNP autoantibodies. The compound mutant mice displayed increased spontaneous and TLR-induced TNFa and IL-6 and B cells from these mice produced much greater levels of these cytokines in response to TLR7 and TLR9. These results suggest that inappropriate B cell survival and persistent inflammation cooperate in the initiation and severity of lupus pathogenesis.

Sylvester Comprehensive Cancer Center, University of Miami, Miami FL
Distinct roles of CD4+ and CD8+ Tregs in regulating human autoreactive T cells, B cells, and antibodies in genetically engineered tonsil organoid system
Authors: Chen Xin , Mallajosyula Venkata , Ghanizada Mustafa , Sola Elsa , Chen Lei , Kamalyan Lilit , Li Jing , Davis Mark M
Published: August 2023

CD4+ and CD8+ regulatory T cells (Treg) are critical for maintaining immune tolerance, and alternations in number and function are associated with detrimental outcomes in infectious and autoimmune diseases. Despite their importance, their distinct roles and mechanisms in regulating self-tolerance need to be better characterized in humans. Here we analyzed the relative contribution of CD4+ and CD8+ Tregs to control autoreactive T cells, B cells, and antibodies in an entirely human tonsil organoid system in vitro. We disrupted their suppressive functions by knocking out FOXP3 and GZMB genes using CRISPR/Cas9 technology. Normally tonsil organoids make robust antibody and cellular responses to live attenuated flu vaccine, but not to autoantigens. In contrast, FOXP3 KO tonsil organoids produced autoantibodies when stimulated with a panel of classical autoantigens, including double-stranded DNA and others. We also detected increased CD8+ T cells specific for autoantigens, including SMCY antigen. With the GZMB KO, we found a marked increase in follicular helper T cells and autoreactive CD4+ and CD8+ T cells. Interestingly the GZMB KO tonsils showed an expansion of plasmablasts, but only a low level of autoantibodies and autoreactive B cells was detected. This indicates that CD8+ Tregs control the early T and B cell activation but not autoantibody production. Moreover, knocking out FOXP3 generated high-affinity HA-specific antibodies in tonsil organoids compared to control, indicating that CD4+ Tregs are a key checkpoint for high-affinity antibodies and the production of autoantibodies. We conclude that CD8+ and CD4+ Tregs have distinct and complementary roles in regulating cellular and humoral responses and preventing autoimmunity.
Eos is a critical transcription factor for T regulatory cell (Treg) function
Authors: Xie Xuan , Tanwar Shalini , Thornton Angela M , Korty Pat , Schwartz Owen M , Smelkinson Margery , Shevach Ethan M
Published: August 2023

Eos is a member of the Ikaros transcription factor family and is highly expressed in Tregs. Mice with a selective deficiency of Eos in Treg develop systemic or organ-specific autoimmune disease at at a young age. We have generated a specific anti-Eos monoclonal antibody. The percentage of Tregs expressing Eos rapidly increases with aging from almost 0% at birth to 30~50% of thymic Tregs and 35~60% of splenic Tregs at 3 weeks of life and then remains stable. Eos is expressed at very low levels on induced Tregs, conventional CD4+ T cells and CD8+ T cells in vivo or in vitro. In the thymus, Eos is only expressed on CD73+ CCR6+ CXCR4+ Tregs, which suggests that Eos+ thymic Tregs represent peripheral Tregs that have recirculated to the thymus. In thymus, Eos expression on Tregs is also highly correlated with CXCR4 and CCR6. Using these markers, we sorted Eos+ and Eos− Tregs and in vitro expanded them using anti-CD3/CD28 beads and IL-2; Eos expression remained stable. Using immunofluorescence microscopy, we have found that Eos is highly co-localized with Helios, another Ikaros family member and HDAC1, but not other Ikaros family members including Ikaros or Aiolos, suggesting that Eos and Helios may form heterodimers and together with HDAC1 collaborate in Treg function. Nevertheless, expression of Eos is independent of Helios and vice versa, as Treg from Helios deficient mice express normal or elevated levels of Eos, and Treg from Eos deficient mice express normal levels of Helios. Taken together, these strongly suggest that Eos plays a related, but distinct role, from Helios in Treg function.

This work was supported by Division of Intramural Research (DIR), NIAID, NIH.
Regulatory T cells limit opportunistic colonization of Staphylococcus aureus to promote lesion resolution in cutaneous leishmaniasis
Authors: Singh Tej Pratap , Amorim Camila Farias , Lovins Victoria , Bradley Charles W , Carvalho Lucas P , Carvalho Edgar M , Grice Elizabeth A , Scott Phillip
Published: August 2023

The severity and responsiveness to treatment in L. braziliensis patients is influenced by a variety of factors, including microbiota and cytolytic T cells. Recently, we studied the skin microbiome in patients and demonstrated that high levels of S. aureus are associated with delayed healing in patients. To understand how S. aureus might influence cutaneous leishmaniasis, we studied the immune responses in mice following colonization with a S. aureus isolate from a L. braziliensis patient. Colonization of the skin with S. aureus alone induced a dominant type-17 response as well as an accumulation of Foxp3 T regulatory cells, and subsequent infection of mice with L. braziliensis led to increased IL-17 dependent pathology. Unexpectedly, transient depletion of total Tregs or RORγtin Foxp3 Tregs in S. aureus colonized mice increased IFN-γ and decreased the IL-17A response in skin. These mice exhibited increased pathology, which was accompanied by epidermal cell death and loss of barrier integrity. Additionally, the mice had substantially more S. aureus in the skin, and in the lymph nodes. In mice colonized with S. aureus and infected with L. braziliensis partial reduction led to a substantial increase in disease and a higher burden of S. aureus. Consistent with these results, in a transcriptional analysis of lesions from L. braziliensis patients, we found that low Foxp3 expression in patients had increased expression of cytolytic genes and IFN-γ, increased S. aureus in the lesions and had a delay in lesion resolution compared to patients with high Foxp3. Taken, together, these results suggest that in L. braziliensis patients Tregs are critical to regulate type-1 responses that in the context of S. aureus can promote more severe disease.
Oxidized phospholipid oxPAPC induces a dysfunctional Th1-like phenotype in regulatory T cells
Authors: Appleton Brenna , Palmer Sydney , Smith Harrison , Stephens Lilly , Major Amy
Published: August 2023

Regulatory T cells (Tregs) are protective in atherosclerosis but numbers are reduced during disease progression due to cell death and loss of stability. Oxidized lipids are abundant in atherosclerotic lesions and can activate innate immune cells, but there is limited information regarding their impact on T cells. Given the Treg loss observed during atherosclerosis progression and increase in oxidized lipid levels in the microenvironment, we sought to determine whether an oxidized phospholipid associated with atherosclerosis, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC), alters Treg differentiation and function. In vitro skews of naïve T cells showed that oxPAPC-treated Tregs were less viable with a greater proportion expressing the Th1 markers T-bet, CXCR3, and IFN-γ. Th1 and Th17 skewed cultures were unaltered by oxPAPC suggesting a Treg specific effect of oxPAPC. OxPAPC-treated Tregs were less suppressive in vitro and were insufficient to inhibit atherosclerosis progression in an adoptive transfer in Ldlr−/− mice fed Western diet for up to 16 weeks. A role for IFN-γ signaling in Treg dysregulation was examined using Treg specific IFNγR1 deficient mice. IFNγR1 deficient CD4+ T cells skewed in the presence of oxPAPC did not adopt the Th1-like Treg phenotype compared to control oxPAPC-treated Tregs, indicating that IFNγR signaling was important for oxPAPC-mediated Treg dysregulation. Bone marrow transplant of Ldlr−/− showed that Treg IFNγR1 signaling was atherogenic in males but protective in females. Overall, our findings demonstrate that oxidized phospholipids directly impact Treg differentiation and decrease their atheroprotective potential.
Effect of malaria parasites’ infection on CD4 cells, haemopoietic cells and haemopoietic factors
Authors: Amadi Emmanuel Chike
Published: August 2023

Malariology Immunity is essential for understanding the pathology, treatment & vaccine production. Changes in CD4, haemopoietic cells & factors (RBC/HB/PCV/MCV/MCH & MCHC) in malaria parasites-infected patients were studied at a teaching hospital in Enugu, Nigeria. 45 patients on doctor’s provisional diagnosis of malaria were examined for Plasmodium infections & the degree of parasitaemia (0/+/++/& +++). Positive (+) & negative (0) samples were thereafter counted for CD4/haemopoietic cells & factors using Flow Cytometry/Automated Blood Cell Counter, respectively. Plasmodium-negative specimens were within reference ranges for CD4 cell count (464–1308); mean value: 835. Fifteen (+) parasitaemia showed lower ranges of CD4 count (502–1282); mean: 678; with immune falls in one (427). Nine from twelve (++) parasitaemia showed crash in CD4 cells counts, range: 301–415; mean: 399. Seven (+++) parasitaemia showed crashes in CD4 cell counts, range: 160–357; mean: 225. Significant rise with increase in parasitaemia occurs in neutrophils and Total WBC. Opposite occurred in the three other primary immune cells & platelets. There were variances in the basic immune cells’ percent & numeric. The haemoglobin factors didn’t exhibited significant change, except platelet that showed marked steady decrease with increase in parasitaemia. Plasmodium infection causes immunosuppression in patients, vivid changes in haemopoietic cells populations/percentages, & platelets. Malaria infection in the immunodeficients patients should accelerate the complications, including death, unlike prevailing reports. Haemolytic anaemia in malaria is likely complication resulting only from chronic cases since there was no vivid anaemia in this study

None