bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of CREB signaling through antagonism of vasoactive intestinal peptide enhances CD8 T cell function in a murine model of AML
Authors: Petersen Christopher T , Li Jian-Ming , Waller Edmund K
Published: January 2023

Signaling pathways that activate CREB may inhibit the potency of T cell responses following stimulation. One of the major mechanisms by which this occurs is the sequestration of CBP, which is also an NF-κB binding partner. One of the major molecules that signals through this pathway is vasoactive intestinal peptide (VIP). VIP is a 28 amino acid peptide that is secreted throughout the body by a variety of cell types including immune cells. VIP has shown to be a potent anti-inflammatory molecule with pleiotropic effects on T cells including cell cycle arrest and inhibition of cytokine secretion. The VIP signaling pathway represents a therapeutic target in disease settings in which a more robust T cell response would be beneficial. Acute myeloid leukemia (AML) is a hematological malignancy that has been explored as a candidate for a variety of T cell-based immunotherapies due to weak CD8 responses to AML blasts. We demonstrate here that administration of a hybrid peptide antagonist that blocks VIP signaling enhances CD8 T cell responses in a murine model of AML. CD8 T cells from treated mice showed enhanced secretion of IFN-γ and TNF-α as well as reduced expression of PD-1. These enhanced effector functions lead to a significant survival enhancement, which was lost when treated mice lacked T cells. Additionally, CD8 T cells from antagonist-treated survivors made up the majority of CD3+ cells following transfer to Rag1 knockout recipients. The phenotype of the cells was consistent with enhanced effector functions as transferred CD8+ T cells were predominately CD44+CD62L−. Taken together, these results demonstrate the potential for VIP antagonism as an immunotherapy strategy in settings of hematological malignancies.
Effector CD8 T cells use different homing receptor/ligand interactions to enter subcutaneous and intraperitoneal B16 melanoma tumors
Authors: Woods Amber N. , Wilson Ashley L. , Srivinasan Nithya , Engelhard Victor H.
Published: January 2023

CD8 T cell infiltration into tumors is critical for controlling cancer and relies on interactions between homing receptors (HR) on CD8 T cells and ligands on the vasculature. However, the ligands expressed on tumor vasculature, and the receptor interactions required for entry into tumors are incompletely characterized. The HR ligands VCAM-1 and E-selectin were highly expressed on the vasculature of B16-F1 melanomas expressing the strong antigen ovalbumin (B16-OVA) and growing subcutaneously but were significantly decreased compared to inflamed skin vasculature. In contrast, the vasculature of intraperitoneal tumors expressed MadCAM-1, negligible E-selectin, and lower VCAM-1. However, MadCAM-1 was not displayed on the luminal surface. B16-F1 parental tumors lacking a strong antigen, and B16-OVA tumors grown in Rag1KO animals, expressed negligible VCAM-1 and MadCAM-1, suggesting that previously infiltrating activated adaptive immune cells drove their expression. VCAM-1 and CXCL9/10 enabled entry of CD8 T cell effectors expressing α4β1+ integrin and CXCR3 into both subcutaneous and peritoneal tumors, while E-Selectin enabled entry of E-Selectin ligand+ effectors preferentially into subcutaneous tumors. MadCAM-1 did not mediate entry of α4β7+ effectors into peritoneal tumors, in keeping with its lack of luminal expression. Disrupting immunosuppression with checkpoint blockade inhibitors may increase HR ligand expression on tumor vasculature and improve CD8 T cell entry into tumors.
Manipulation of IL-2 signals by IL-2/antibody complex and CD25 blockade improves tumor immunity, reprograms regulatory T cells, and augments CD8+ central memory in an ovarian cancer model
Authors: Drerup Justin Michael , Padron Alvaro Souto , Chen Wanjiao , Clark Curtis Anthony , Curiel Tyler Jay
Published: January 2023

We used a defined IL-2/αIL-2 complex that directs IL-2 to stimulate only intermediate-affinity IL-2 receptor (CD122/132) to promote tumor rejection and avoid regulatory T cell (Treg) expansion. However, it is unknown if IL-2/αIL-2 alters Treg function, as Tregs also express CD122/132. After challenge with ID8agg ovarian cancer (OC) cells, IL-2/αIL-2 greatly reduced tumor burden, and increased IFN-γ, TNF-α, CD44, and CD25 in CD4+ non-Tregs and CD8+ T cells, as expected. IL-2/αIL-2 was clinically effective, yet it lowered the ascites CD8+/FoxP3+ Treg ratio and increased per cell FoxP3 levels in Tregs, suggesting a change in Treg function. IL-2/αIL-2 decreased the ratio of CD62L+ central Tregs to CD44+ effector Tregs in ID8agg ascites, indicating modulation of Treg differentiation. Surprisingly, ascites Tregs produced IFN-γ, IL-2, and TNF-α in control-treated OC mice, which was abolished by IL-2/αIL-2, suggesting further alteration of Treg differentiation and function. CD25 (high-affinity IL-2 receptor subunit) expression on Tregs was reduced by IL-2/αIL-2, which could lower Treg IL-2 sequestration and increase IL-2 available to antitumor T cells. CD25 blockade with αCD25 during IL-2/αIL-2 treatment reduced clinical efficacy but greatly increased central memory (CM; CD62L+CD44+) CD8+ T cells in blood and mesenteric (tumor-draining) lymph nodes. Thus, reduction of tumor burden by IL-2/αIL-2 is associated with reprogramming of Treg differentiation and function in an OC model. CD25 blockade after IL-2/αIL-2 could improve response durability by boosting CM CD8+ formation. These data demonstrate novel effects of IL-2-modulating drugs on Tregs and CD8+ memory and will aid development of more effective anticancer immunotherapy.
Impact of hypoxia on IFNγ-dependent responses: implications for radiotherapy and anti-tumor immunity
Authors: Murthy Aditi , Gerber Scott A , Koch Cameron J , Lord Edith M
Published: January 2023

Tumor hypoxia occurs due to an increase in demand for oxygen by the rapidly growing tumor cells together with reduction in the supply of oxygen due to malformed and non-functional tumor vasculature. Tumor hypoxia offers resistance to radiotherapy (RT). Interestingly, a new paradigm has emerged suggesting that hypoxia may also suppress immunotherapy, however the mechanisms behind this observation remain undetermined. Our laboratory and others have shown that IFNγ conditions the tumor milieu and is important for the efficacy of RT. As a result, we hypothesized that hypoxia could inhibit IFNγ mediated anti-tumor responses resulting in decreased RT efficacy. Hypoxia could be modulating the production of intratumoral IFNγ, and/or reducing the ability of cells to respond to IFNγ. To test this we utilized Colon38, a murine tumor model, and measured intratumoral hypoxia using the hypoxia marker EF5 and demonstrated a time dependent increase of hypoxia within untreated tumors. Irradiation resulted in a decrease in total tumor hypoxia. We also detected various sub-populations of hypoxic immune cells in both the untreated and irradiated tumors, with macrophages being the majority population. Importantly, CD8+ T cells, which are an important source of IFNγ and mediate effector anti-tumor responses, were present in these tumors but were not co-localized with hypoxic regions. This deficiency of effector T cells in the hypoxic regions indicates that tumor cells in these areas may also escape from immunotherapy. To alleviate this, we are examining if local IL-12 delivery, which normalizes tumor vasculature, can decrease hypoxia thus increasing IFNγ levels and responsiveness and ultimately enhancing tumor control. Funded by NIH R01 CA28332.
Dicer regulates tumor immunogenicity in melanoma
Authors: Hoffend Nicholas C , Magner William J , Tomasi Thomas B
Published: January 2023

Immunotherapeutic strategies in cancer rely upon tumor immunogenicity for their efficacy. However, the regulation of immune effector molecules in melanoma is poorly understood. High Dicer expression in melanoma correlates with more aggressive tumors and poor prognosis/patient survival. Dicer regulates gene expression through processing of microRNAs, but its potential role in regulating the immunogenicity of melanoma has not been described. Here, we demonstrate that Dicer suppresses MHC class I and II cell surface expression, as well as CD40 and PD-L1 in melanoma. By using a genetic approach wherein we challenged C57BL/6 mice with melanoma cells stably expressing Dicer shRNA, we showed that Dicer knockdown decreased tumor progression and extended overall survival. Interestingly, Dicer knockdown had no effect on melanoma cell proliferation or survival in vitro, suggesting that the delay in tumor growth may be due to an augmented immune response. Tumor control of Dicer knockdown melanoma cells was dependent upon an intact immune system, as revealed by studies using NSG mice. Mechanistically, this anti-tumor immune response was dependent upon CD8+ T cells. Subsequent studies demonstrated that melanoma Dicer expression was able to suppress CD8+ T cell killing in vitro. Taken together, this suggests a model wherein Dicer negatively regulates melanoma immunogenicity and protects against CD8+ T cell anti-tumor immunity. Using this insight, we may better stratify patients for immunotherapies such as checkpoint inhibition and cell-based vaccination strategies. Dicer also makes an attractive target for modulating baseline tumor cell immunogenicity, subsequently enhancing melanoma immunotherapy and patient survival.
Development of a Novel Preclinical Model Based on HLA-A2 Transgenic Mice to Evaluate T-Cell Receptor Mimic Monoclonal Antibodies as Therapeutic Agent against Brain Metastases of Human Breast Cancer
Authors: Park Jee Hyun , Yang Li , Alqahtani Faleh , Bickel Ulrich
Published: January 2023

The HLA-A2 restricted T-cell receptor mimic (TCRm) antibody RL6A has been shown to have antitumor effects in a breast cancer orthotopic xenograft model with MDA-MB-231 cells. Transgenic mice expressing HLA-A2 also take up RL6A into brain across the blood-brain barrier (BBB). The present study evaluates different approaches to obtain a reliable brain tumor model using HLA-A2 transgenic mice, in which pharmacokinetics and antitumor efficacy of RL6A on brain metastasis could be studied. First, available immunocompromised HLA-A2 transgenic strains, such as the HLA-A2 transgenic NSG mouse (Jackson Labs), were utilized and various tumor injection methods were tested. As NSG mice are the most immunocompromised and vulnerable strains, intracardiac injection of brain-seeking human breast cancer cells invariably resulted in tumors of multiple peripheral organs even when using a brain seeking subclone of MDA-MB-231. More targeted approaches (common carotid artery or retrograde external carotid injections) reduced the tumor burden in peripheral organs intracardiac injection, but turned out being too invasive procedures for this brittle mouse strain. Stereotaxic intrastriatal injection in NSG mice resulted in tumor growth at the implantation site within brain, but also in skull bone and dura. Therefore, the NSG background appeared unsuitable for our purpose. We then evaluated intracardiac injections of tumor cells in a Foxn1nu nude mouse strain on a C57BL/6 background, which developed predominantly brain tumors and no tumors in major peripheral organs (lung, liver, kidney). Cross-breeding with C57BL/6 transgenic HLA-A2 mice was initiated and yielded a novel HLA-A2 transgenic nude mouse strain.
Human anti-MUC1 antibodies elicited by a prophylactic cancer vaccine for CAR T cell immunotherapy.
Authors: Lomueller Jason Jakob , Sato Shuji , Popova Lana , Chu Isabel M , Tucker Meghan A , Barberena Roberto , Innocenti Gregory M , Cudic Mare , Ham James D , Cheung Wan Cheung , Polakiewicz Roberto D , Finn Olivera J
Published: January 2023

Hypoglycosylated MUC1 is a tumor-associated protein that is expressed on over 80% of all human cancers including adenocarcinomas of the colon, pancreas, breast, lung, prostate, and ovary, and multiple myeloma. In our recent clinical trial, individuals at-risk for colon adenocarcinoma received a prophylactic MUC1 cancer vaccine. Many individuals responded producing high titers of anti-MUC1 IgG antibodies with no detectable toxicity. This trial provided a rare source of human antibodies elicited and affinity-matured in a healthy human host to abnormal MUC1. Using our recently developed proteomics method, we isolated and identified 13 anti-MUC1 antibodies representing 7 different clonotypes. These antibodies bind to several different epitopes on the MUC1 vaccine peptide with a range of affinities (15.7μM to 130pM). They also stain MUC1 on human cancer cell lines and colon, breast, lung, and pancreas adenocarcinoma tissue sections while showing no reactivity against a large panel of normal tissues that express MUC1. We constructed lentiviral vectors encoding chimeric antigen receptors (CARs) using scFv’s of several of the antibodies as antigen binding regions and a variety of co-signaling domain architectures. Several of the scFv’s were able to retarget human primary T cells to become activated and produce cytokines in a MUC1-dependent manner and to lyse a variety of MUC1+ human tumor cell lines. Preclinical testing in mouse tumor xenograftgrafts is underway. Being of fully human origin and showing a high-degree of tumor specificity and efficacy in preclinical experiments, these antibodies will be tested in future clinical trials for potential approval for therapy of cancer patients.
Differential kinetics of CD4+ and CD8+ T cell responses: implications for the assessment of immune responses
Authors: Hokey David A , Graves Andrew J , O’Dee Dawn M , Higgins Adam , Hooper Idris , Schaeffer Jacqueline , Walker Barry
Published: January 2023

Intracellular cytokine staining (ICS) to detect cellular immune responses to infection or vaccination has become routine. Laboratories develop and optimize ICS assays independently and there are many variations of the assay in use today. However, little is known regarding the underlying biology that results in the variable outcomes following assay optimization. The results from the different assays can range from highly similar (slight changes in magnitude) to incredibly different, such as large changes in magnitude or a change in the responding cell types detected. These large differences prompted our investigation into the underlying reasons for the discrepancies in reported data for clinical trials. Here, we used leukapheresis samples from CMV-reactive volunteers to examine viability over time, the effect of costimulation, and response kinetics. The data show that golgi transport inhibitors diminish cell viability after 12 hours of stimulation, emphasizing the importance of viability markers particularly during long stimulations. Costimulatory antibodies increase responses but at the expense of sensitivity, given the increase in background. Interestingly, we found that CD4+ and CD8+ T cells exhibit differential kinetics in their response to CMVpp65 peptides. CD8+ T cells were largely exhausted by 6 hours following the initiation of stimulation, while CD4+ T cells continue to make cytokines well past 20 hours of stimulation. These data have implications on the design of ICS assays and the interpretation of data generated by ICS. Furthermore, the data provide a basis for understanding the differences in ICS assays and provide insight into the kinetics of CD4+ and CD8+ T cell responses.
Tumor blood vessel-associated antigens, DLK-1 and DLK-2, as targets for anti-cancer immunotherapy
Authors: Fabian Kellsye Paula , Storkus Walter
Published: January 2023

Tumors induce pathological angiogenesis that results in a tumor vascular network that is highly abnormal. These aberrant blood vessels support tumor growth and progression and also immunosuppression by promoting a hypoxic and high interstitial fluid pressure-environment that impedes immune trafficking to the tumor bed. Hence, normalization of tumor blood vessels is essential in ensuring success of anti-cancer therapy. We have previously shown that pericytes in tumor blood vessels of murine renal cell carcinoma (RENCA) express the Notch antagonist delta-like homolog-1 (DLK1). DLK1-targeted vaccination of RENCA-bearing mice resulted in vascular normalization and CD8+ T cell-dependent inhibition of tumor growth. Here we show that DLK2, a homolog of DLK1, is upregulated in RENCA pericytes after DLK-1 vaccine-induced removal of DLK1 in the tumor microenvironment (TME). Using recombinant lentiviruses and adenoviruses encoding full-length DLK1 and DLK2, we demonstrate that coordinate vaccination against these two antigens leads to superior antigen-specific CD8+ T cell-induced tumor growth inhibition than vaccination with either antigen alone. We also show that DLK-1 and DLK-2 coordinate vaccination shifts the TME to a more “immunocompetent” phenotype by normalizing tumor vasculature and decreasing the immunosuppressive cell population. We conclude that specific vaccination targeting DLK-1 and DLK-2 is a promising approach for the treatment of vascularized tumors.
Toll-like receptor ligands in conjunction with agonist anti-OX40 mAb immunotherapy possess differential capacity to revive anergic self-reactive CD8 T cells
Authors: Kasiewicz Melissa J. , Farhad Mohammad , Redmond William L.
Published: January 2023

Previous work has shown that the induction of CD8 T cell anergy to a prostate-specific self-antigen can be reversed following ligation of the TNFR family member OX40 (CD134) with an agonist anti-OX40 mAb plus vaccination. The provision of the Toll-like receptor 4 (TLR4) ligand bacterial lipopolysaccharide (LPS) further enhanced T cell responses following combined anti-OX40/vaccine immunotherapy. Although this approach facilitated increased CD8 T cell infiltration into the prostate, these cells were unable to elicit significant destruction of the prostate tissue. In the current study, we examined the extent to which co-administration of select TLR ligands would affect the ability of anti-OX40/vaccine therapy to revive prostate-specific CD8 T cells rendered anergic in vivo. These data revealed a differential capability of TLR agonists in conjunction with anti-OX40/vaccine immunotherapy to generate robust effector CD8 T cells capable of trafficking into normal prostate epithelia and inducing tissue-specific damage. These data provide the framework for translational studies to evaluate the efficacy of administering OX40 agonists with tumor-specific vaccination and TLR ligand adjuvants to maximize T cell-mediated therapeutic responses.
Efficacy of an oral recombinant attennated Salmonella vaccine encoding cjaA in turkeys colonized with Campylobacter jejuni
Authors: Sylte Matthew Jon , Looft Torey P. , Chandra Lawrance C. , Bearson Shawn M. , Bearson Bradely L. , Susta Leonardo
Published: January 2023

Campylobacter jejuni spp. jejuni, (C. jejuni) is a commensal bacterium in the lower gastrointestinal tract of poultry (e.g., chickens and turkeys), where it causes no clinical disease. Consumption of C. jejuni-contaminated poultry products is the most likely route of transmission to humans. Strategies previously attempted in chickens to reduce intestinal C. jejuni colonization include administration of probiotics, non-antibiotic feed additives, vaccines and bacteriophages. These interventions have not been evaluated in turkeys. Recombinant attenuated Salmonella vaccines (RASV) encoding C. jejuni cjaA are reported to reduce cecal C. jejuni colonization in chickens. The goal of this study was to evaluate the efficacy of a RASV encoding cjaA in turkey poults experimentally colonized with C. jejuni. The cjaA gene from C. jejuni was cloned into RASV strain χ8133 (RASV-cjaA). A control RASV χ8133 (RASV-control) was generated lacking a cjaA insert. At different dates of age, poults received either one or two doses of RASV-cjaA. Other poults received 2 doses of RASV-control. Intestinal colonization by RASV-cjaA was evaluated and was detectable up to 6 days after oral inoculation. Poults were orally challenged with C. jejuni and euthanized at days 3, 7, 14 and 28 days post-challenge to evaluate cecal colonization (cfu/g content) and histopathological changes. Poults vaccinated with 2 doses of RASV-cjaA showed significant reduction (P<0.01) in C. jejuni cecal colonization and histopathological lesion scoring, as compared to those receiving a single dose of RASV-cjaA or RASV-control. These data demonstrate that oral RASV-cjaA significantly reduced C. jejuni colonization in turkeys, which may enhance food safety.
The role of IRF7 and NF-κB pathways in the induction of antiviral responses in chicken tracheal epithelial cells following exposure to TLR3 and 4 ligands
Authors: Barjesteh Neda , Abdelaziz Khaled Taha , Sharif Shayan
Published: January 2023

With recent outbreaks of avian influenza viruses (AIV), there is a need for better understanding of host responses in the respiratory system to AIV in order to develop and improve prophylactic procedures for control of AIV. Toll-like receptors (TLRs) are molecules in the immune system that rapidly induce antiviral responses in the host. Activation of TLRs by their ligands leads to a variety of cellular responses, including the production of interferons (IFNs). However, there is little information about induced antiviral responses in chicken trachea following TLR ligand treatment. We cultured primary chicken tracheal epithelial cells (cTECs) to determine antiviral responses induced by TLR3 and 4 ligands. These cells were treated with polyI:C and LPS from Escherichia coli 026:B6, TLR3 and 4 ligands respectively, before AIV infection. We also used BX795 and celastrol, inhibitors of IRF7 and NF-κB pathways, respectively, in order to gain insight into the mechanisms involved in the induced antiviral responses. Our results showed that treatment of cTECs with TLR ligands reduced the replication of AIV in cTECs. Treatment of cTECs with polyI:C resulted in highest reduction of viral replication in cTECs followed by treatment of cTECs with LPS. BX795 increased AIV replication in polyI:C and LPS treated cells while celastrol increased AIV replication in polyI:C treated cells. Overall, these results provided evidence that cTECs are able to mount antiviral responses against AIV after TLR ligand stimulation. Moreover, we determined the role of IRF7 and NF-κB signalling pathways in the induction of antiviral responses. Studies are underway to examine the ability of cTECs to produce type I IFNs and the effect of other cells, such as macrophages, on cTECs.
Host genetic factors and miRNA-linked dendritic cell responses associated with the outcome of treatment response in HIV-1/HCV co-infected individuals
Authors: Jain Pooja , Sehgal Mohit , Narang Rubina , Ginwala Rashida , Zeremski Marija , Talal Andrew , Elrod Elizabeth , Grakoui Arash , Li Qi-Ging , Philip Ramila , Khan Zafar
Published: January 2023

HIV-1/HCV co-infection is a significant burden on global economy and public health. PEGylated interferon and ribavarin remain the essential components of anti-HCV treatment. We investigated the host genetic and immunological correlates of successful treatment response in conjunction with mechanisms by which PEG-IFN is able to clear the virus in a cohort of HIV-1/HCV co-infected individuals undergoing IFN/RBV treatment. First we demonstrate that functional state of DCs before/during therapy influences the treatment outcome. Upon genotyping IFNL3 polymorphisms rs12979860, rs4803217 and ss469415590, we found rs12979860 to be a better predictor of treatment outcome. Next, we compared the expression of forty-six IFN-stimulated genes prior to and after the treatment and observed that pre-treatment levels of several ISGs were higher in SVRs compared to NRs. In continuation, we identified miRNAs whose expression can be regulated by IFN in periphery. miRNA expression patterns in mDCs and pDCs in response to IFN-α were examined and observed miR-221 downregulation via IFN induced STAT3 inhibition in both. Using in-silico approaches followed by experimental validation, CDKN1C, CD54 and SOCS1 were identified as miR-221 targets. Moreover, miR-221 overexpression in mDCs enhanced their secretion of proinflammatory cytokines IL-6 and TNF-α but reduced the secretion of anti-inflammatory cytokine IL-10. These observations were extended and correlated with those obtained with patients’ PBMCs, hepatocytes and kupffer cells from HCV infected individuals as well as individuals with alcoholic cirrhosis. In summary, these studies demonstrated the role of IFN-α/miR-221 axis in HCV pathogenesis and response to IFN-based treatments.
The survival of monocytes infected by human cytomegalovirus is dependent on a unique signalsome induced during entry.
Authors: Peppenelli Megan A , Arend Kyle C , Cojohari Olesea , Moorman Nathaniel J , Chan Gary C
Published: January 2023

HCMV infection of immunocompromised individuals often leads to multi-system organ failure. The development of multi-system organ failure is dependent on the ability of HCMV to spread to peripheral organs, which is mediated by blood monocytes. We have previously shown HCMV to extend the short 48hr lifespan of monocytes. Mechanistically, HCMV upregulated cellular myeloid leukemia sequence 1 (Mcl-1) and heat shock protein 27 (HSP27), to block the proteolytic cleavages necessary for the formation of active caspase 3. We now show that, compared to other myeloid survival factors, HCMV infection more efficiently upregulates Mcl-1 and the only survival factor to rapidly induce HSP27. These observations, suggest a unique virus specific mechanism of induction; thus, we examined receptor: ligand signaling events, since viral anti-apoptotic proteins are not expressed until differentiation into macrophages is complete. We determined that HCMV-induced EGFR/PI3K/AKT signaling increased the transcription of both Mcl-1 and HSP27. Yet, protein expression of Mcl-1 and HSP27 is increased via the gH/aVb3/src and the gB/EGFR pathways, respectively. To address this discrepancy between transcripts and protein we evaluated the effects of HCMV on the translation of Mcl-1 and HSP27. Indeed, we found, in contrast to myeloid survival factors, HCMV stimulated the translation of Mcl-1 and HSP27. Overall, these data indicate that inhibition of apoptosis by HCMV is through the unique signaling regulation of Mcl-1 and HSP27; ensuring short-lived monocyte survival past the normal 48hr viability checkpoint, a key event necessary for viral dissemination.
Transcriptional regulation in the development of CD8ab+ iNKT cells in a humanized mouse model
Authors: Yuan Weiming , Wen Xiangshu , Li Michelle , Xiong Ran , Bosselut Remy , Rao Ping
Published: January 2023

Invariant NKT (iNKT) cells are innate-like T cells showing potent anti-tumor function in conventional mouse models. In sharp contrast, iNKT cell ligands have had limited efficacy in human anti-tumor clinics, mostly due to the profound differences in the properties and compositions of iNKT cells between human and mice, in particular, the presence of CD8+ iNKT cells only in humans. To build more relevant in vivo models for studying human iNKT cells, we first developed a CD1d-humanized mouse model (hCD1d-KI) with human CD1d knocked in (Proc. Natl. Acad. Aci. USA 110: 2963–8, 2013). To further humanize the mouse model, we introduced human invariant TCRa chain (Va24Ja18) into the hCD1d-KI mice. Interestingly, we observed a substantial subset of iNKT cells expressing CD8ab in the new humanized mouse model (J. Immunol. 195:1459–69, 2015). The CD8ab+ iNKT cells show a strong Th1-biased cytokine response and potent cytotoxicity upon activation. The low binding of iNKT TCRs to human CD1d/lipid complex and high prevalence of Vb7 TCRb among these CD8+ iNKT cells strongly suggested a novel low avidity-based developmental program for the ontogeny of these iNKT cells which includes the suppression of the transcriptional factor, Th-POK. This suppression of Th-POK is essential for the development of CD8ab+ iNKT cells as these iNKT cells can not be detected upon forced expression of Th-POK early in T cell development. Our establishment of the new mouse model extensively humanized in CD1d/iNKT TCR system will greatly facilitate the investigation of in vivo functional properties of human iNKT cells as well as future design and optimization of iNKT cell ligands for diverse immunotherapies.
The Dendritic Cell MHC II Peptidome Derives from a Variety of Processing Pathways and Includes Peptides with a Broad Spectrum of HLA-DM Sensitivity
Authors: Stern Lawrence , Santambrogio Laura
Published: January 2023

The repertoire of peptides displayed in vivo by MHC II molecules derives from a wide spectrum of proteins produced by different cell types. While intracellular endosomal processing in dendritic cells and B cells has been characterized for few antigens the overall range of processing pathways responsible for generating the MHCII peptidome are currently unclear. To determine the contribution of non-endosomal processing pathways we eluted and sequenced over 3000 HLA-DR1-bound peptides presented in vivo by dendritic cells. Processing enzymes were identified by reference to a database of experimentally determined cleavage sites and experimentally validated for four epitopes derived from complement 3, collagen II thymosin b4 and gelsolin. We determined that self-antigens processed by tissue-specific proteases, including complement, matrix metalloproteases, caspases, and granzymes, and carried by lymph, contribute significantly to the MHC II self-peptidome presented by conventional dendritic cells in vivo. Additionally, presented peptides exhibited a wide spectrum of binding affinity and HLA-DM susceptibility. The results indicate that the HLA-DR1-restricted self-peptidome presented under physiological conditions derives from a variety of processing pathways. Non-endosomal processing enzymes add to the number of epitopes cleaved by cathepsins, altogether generating a wider peptide repertoire. Together with HLA-DM-dependent and HLA-DM-independent loading pathways, this ensures that a broad self-peptidome is presented by dendritic cells.
MHCII with full peptide occupancy is a substrate for HLA-DM catalyzed peptide exchange
Authors: Reyes-Vargas Eduardo , Barker Adam P , Zhou Zemin , He Xiao , Jensen Peter E
Published: January 2023

The major histocompatibility complex class II (MHCII) antigen-processing pathway is critical for adaptive immunity as the mechanism through which ligands are generated for recognition by CD4 T cells. HLA-DM edits the repertoire of peptides that are presented to T cells by catalyzing multiple rounds of peptide exchange on MHCII molecules. The catalytic potency of DM varies such that peptide exchange is markedly enhanced on some peptide-MHC (pMHC) complexes and not others. This capacity to differentially edit complexes influences specificity and immunodominance in T cell responses. The catalytic mechanism and rules that govern DM susceptibility are not fully understood. Evidence suggests that partial occupancy at the N-terminus of bound peptides is critical for complex sensitivity to DM. We employed a fluorescence polarization assay to independently assess the effective affinity and catalytic turnover of DM, components of the catalytic mechanism, for pMHC complexes in real-time kinetic measurements of DM activity. We found that a complex with an N-terminal truncated form of the immunodominate HA peptide, resulting in the loss of three H-bonds mediated by the P-2 and P-1 residues, showed an expected decrease in complex stability and increase in affinity for DM binding, but an unexpected increase in the catalytic turnover when compared to the unmodified HA-MHC complex. This result was even more pronounced when a conserved bidentate H-bonding residue near the P1 anchor was disrupted by site-directed mutagenesis on an HA-MHC complex. Together, this implies that pMHC interactions near the N-terminal segment of a peptide may be intact in the functionally active DM-pMHC catalytic complex and provide insights into the DM catalytic mechanism.
Targeting of B cell class-switching and plasma cell survival pathways by a Rab7 inhibitor suppresses autoantibody responses in murine lupus
Authors: Lou Zheng , Lam Tonika , Kulp Dennis , Wang Rui , Taylor Julia , Casali Paolo , Xu Zhenming
Published: January 2023

B cell class switch DNA recombination (CSR) and generation/maintenance of plasma cells underpin the production of pathogenic IgG autoantibodies in lupus. To address whether these two B cell lineage-specific processes can be targeted to inhibit the autoantibody response in lupus, we used a small molecule compound to specifically block the activity of the small GTPase GTPase, as prompted by our previous findings that Rab7 plays a B cell-intrinsic role in antibody responses. Inhibition of Rab7, which was upregulated in lupus B cells, suppressed pathogenic autoantibody production, arrested disease development and extended the life-span in MRL/Fas and Sle1/Sle2/Sle3 lupus-prone mice. The reduction in class-switched autoantibody response occurred despite normal numbers and functions of T cells, dendritic cells and macrophages, but was associated with significantly decreased numbers of IgG-expressing B cells and plasma cells. Accordingly, B cell class-switching and plasma cell survival, but not B cell differentiation into plasma cells, were inhibited by the Rab7 inhibitor through suppression of NF-kB activation, as revealed by impairment in NF-kB-dependent gene expression. Conversely, CSR and plasma cell survival could be rescued by an IKKb constitutively active mutant. These findings suggest a small molecule that targets two important B cell differentiation stages to effectively blunt autoantibody responses in lupus.

Supported by NIH grants AI 079705 and AI 105813 and the Alliance for Lupus Research (ALR) grant ALR 295955 to P.C.
Endogenous dsRNA and dsDNA sensing is increased in transitional B cells in BXD2 autoimmune-prone mice
Authors: Mountz John D , Hamilton Jennie Ann , Wu Qi , Yang PingAr , Luo Bao , Liu Shanrun , Li Jun , Hsu Hui-Chen
Published: January 2023

Enhanced nucleic acid-sensing (NS) pathways has been associated with development of autoimmune disease. Increased type I IFN signaling is identified in lupus prone BXD2 mouse B cells and there is a predominant production of autoantibodies against RNA binding autoantigens in BXD2 mice. Determination of the expression of Usp18 and Oasl2 in early CD93+ transitional B cells (T1 and T2) revealed a surprising finding that early transitional B cell exhibited elevated type I IFN signature gene expression, compared to CD93− mature B cells. Consistent with this, there is also elevated expression of NS pathway genes in early transitional B cells. The most highly expressed were the dsRNA sensing Rig1 and transcription factors Irf3 and Irf7, which were expressed at higher levels compared to pDCs. Interestingly, although Usp18 and Oasl2 levels were undetectable in BXD2-Ifnar−/− transitional B cells, dsRNA sensing gene expression were highly expressed suggesting an IFNAR-independent up-regulation of dsRNA NS pathway in transitional B cells. There was lower expression of Mda5 and Pkr5, and genes involved in DNA sensing including Zbp1 and Irf9. Stimulation of B cells with a RIG1 agonist, poly(I:C)-LMW/LyoVec leads to significantly elevated induction of NS pathway genes in both WT and IFNAR deficient transitional B cells from BXD2 mice. These results suggest that transitional B cells have an intrinsic enhanced nucleic acid-sensing pathway, allowing them to rapidly react to nucleic acid stimulation. Dysregulation of this pathway as a result of apoptotic cell clearance defects can act together with type I IFN stimulation to lead to survival and maturation of B cells that by-pass tolerance checkpoint 2 and produce anti-nucleic acid autoantibodies in lupus.
B cell IFN-γ receptor signaling promotes autoimmune germinal centers via cell-intrinsic induction of BCL-6
Authors: Jackson Shaun W , Jacobs Holly , Arkatkar Tanvi , Dam Elizabeth M , Scharping Nicole , Kolhatkar Nikita , Hou Baidong , Buckner Jane H , Rawlings David J
Published: January 2023

Dysregulated germinal center (GC) responses are implicated in the pathogenesis of human autoimmune diseases, including systemic lupus erythematosus (SLE). Although type 1 interferons (IFNs) are most frequently associated with lupus pathogenesis, type 2 interferon (IFN-γ) has also been shown to promote SLE. However, the respective impact of these cytokines in promoting B cell activation during humoral autoimmunity has not been addressed. Here, using a chimeric model of B cell-driven autoimmunity, we demonstrate that B cell type 1 IFN receptor signals accelerate, but are not required for lupus development. In contrast, B cells functioning as antigen presenting cells initiate CD4+ T cell activation via B cell MHCII expression, resulting in prominent T cell IFN-γ production. Strikingly, B cell-intrinsic deletion of the IFN-γ receptor was sufficient to abrogate autoimmune GCs, class-switched autoantibodies and systemic autoimmunity. Mechanistically, although IFN-γ receptor signals increased B cell T-bet expression, B cell-intrinsic deletion of T-bet exerted an isolated impact on class-switch recombination to pathogenic IgG2c autoantibody subclasses without impacting GC development. Rather, in both murine and human B cells, IFN-γ synergized with BCR, TLR and/or CD40 activation signals to promote cell-intrinsic expression of the GC master transcription factor, BCL-6. Finally, IFN-γ driven BCL-6 expression in B cells was blocked using clinically-relevant Janus kinase inhibitors, ruxolitinib and tofacitinib. Our combined findings identify a novel B cell-intrinsic mechanism whereby interferon signals promote lupus pathogenesis, implicating this pathway as a potential therapeutic target in SLE.
Follicular T helper cells and T helper 17 cells cooperate in promoting B-cell responses in neuro-inflammation
Authors: Quinn James L , Agasing Agnieshka , Ko Rose , Axtell Robert C.
Published: January 2023

Both T-cells and B-cells are implicated in the pathology of multiple sclerosis, but how these cells cooperate to drive disease activity is still unclear. Recent studies using experimental autoimmune encephalomyelitis (EAE) demonstrated that the T helper 17 (TH17) pathway is correlated with increased numbers of ectopic B-cell follicles in the CNS. As follicular T helper (TFH) cells are mediators of B-cell response, our hypothesis was that TH17 cells and TFH cells cooperate to drive pathogenic B-cell responses in the CNS and contribute to the severity of EAE. Therefore, the main objective of this project was to examine the role of TFHs in EAE induced by the transfer of myelin-specific TH17s (TH17 EAE).

At the peak of TH17 EAE, we saw 5.6–18.6% of CD4 cells in the CNS were CXCR5+PD1+ TFHs. Furthermore, the number of TFHs in the CNS was strongly correlated with the number of infiltrating B-cells and disease severity. We next transferred TH17s from congenic CD45.1 mice into CD45.2 recipient mice to determine whether the TFH cells were donor or recipient derived. We observed that the TFHs were CD45.2 recipient-derived whereas IL-17+ cells were CD45.1 donor-derived. We next assessed whether myelin-specific TFHs transferred into recipient mice were capable of migrating to the CNS and causing EAE. We found that TFHs transferred alone failed to traffic to the CNS. However, when TFHs were co-transferred with TH17s into recipient mice, we found increased numbers of TFHs in the CNS compared to the CNS of mice transferred with only TH17s.

These results show that TH17s migrate to the CNS to initiate neuro-inflammation in EAE and mediate of a second wave of CNS-infiltrating TFHs that facilitate a B-cell response in the CNS and contributes to disease severity.
Bim is a candidate gene in the regulation of merocytic dendritic cell proportion
Authors: Audiger Cindy , Chabot-Roy Geneviève , Lesage Sylvie
Published: January 2023

In contrast to conventional dendritic cells (cDC), when merocytic DC (mcDC) present antigens (Ag) derived from apoptotic bodies, T cell anergy is reversed rather than induced. Although helpful to tumour clearance, reversing T cell anergy is detrimental in autoimmunity. Interestingly, mcDC are present in higher proportion in type 1 diabetes (T1D)-prone NOD mice than in B6 mice. Recently, we found that the Idd13 T1D locus is linked to the control of mcDC number. Indeed, NOD.B6-Idd13 congenic mice are resistant to T1D and have the same low number of mcDC as the parental B6 mice. Thus, we hypothesize that defining the genetic factor implicated in the regulation of mcDC number could help maintain self-tolerance and prevent T1D onset. Within the Idd13 locus, both B2m and Bim are likely candidate genes. By exploiting both B2m−/−and Bim−/−mice, we show that B2m does not affect mcDC number while mcDC were significantly increased in Bim−/−mice. Competitive hematopoietic chimera validate Bim as an intrinsic factor implicated in the regulation of mcDC proportion. In line with a role for Bim in regulating mcDC number, we demonstrate that the caspase activity and Bcl2 expression is not differentially expressed between the different mouse model, suggesting that Bim is implicated in mcDC proportion regulation via a caspase-independent pathway.

Together, these data demonstrate that Bim, encoded within the Idd13 locus on the chromosome 2 modulates the number of mcDC. Identifying factors that facilitate apoptosis of mcDC may help prevent autoimmunity. Still, whether restoring Bim expression in NOD mice would prevent T1D remains to be addressed.
BCAP promotes TLR-induced IFNα in plasmacytoid DC
Authors: Chu Talyn , Hamerman Jessica
Published: January 2023

Systemic lupus erythematosus (SLE) severity is correlated with elevated serum levels of type I interferons (IFN), specifically IFNα. pDC are important in the pathogenesis and etiology of SLE due to their ability to produce large amounts of IFNα. TLR-induced INFα production in pDC occurs upon RNA or DNA recognition by TLR7 or 9 respectively. Two events are necessary to induce IFNα by pDC: 1) the binding of ligand to TLR7 or TLR9 within the early endosome, and 2) the phosphorylation and nuclear translocation of the transcription factor IRF7. TLR7 and TLR9-induced IRF7 translocation to the nucleus and subsequent IFN production by pDC is dependent on phosphatidylinositol-3 kinase (PI3K), but how PI3K regulates this process remains undefined. The PI3K pathway is important for many cellular processes, but it is unknown how PI3K activation is induced by TLR signaling in pDC. Our lab showed that the cytosolic signaling adapter B cell adaptor for PI3K (BCAP) links TLRs to PI3K activation in macrophages, resulting in inhibition of TLR-induced inflammatory cytokines. In contrast, here we show BCAP promotes TLR7 and TLR9-induced type I IFN production in pDC. We found that BCAP was required for optimal TLR9 and TLR7-induced IFNα production in pDC grown from BM in vitro in presence of Flt3L or sorted directly ex vivo, while inflammatory cytokine levels were less affected. Strikingly, BCAP−/− mice produced much less serum IFNα early after injection of TLR9 agonist than WT mice, consistent with a pDC IFNα defect. BCAP−/−pDC have reduced AKT phosphorylation when stimulated with TLR agonists, showing BCAP activates PI3K downstream of TLR in pDC. Overall, we show a novel role for BCAP in regulating IFNα production in pDC.
A critical link between Lyn-mediated suppression of the TLR-MyD88-IRF5 pathway and the development of SLE-like disease
Authors: Ban Tatsuma , Sato Go , Nishiyama Akira , Akiyama Ai , Takasuna Marie , Umehara Marina , Suzuki Shinsuke , Ichino Motohide , Matsunaga Satoko , Kimura Ayuko , Kimura Yayoi , Yanai Hideyuki , Miyashita Sadakazu , Kuromitsu Junro , Tsukahara Kappei , Yoshimatsu Kentaro , Endo Itaru , Yamamoto Tadashi , Hirano Hisashi , Ryo Akihide , Taniguchi Tadatsugu , Tamura Tomohiko
Published: January 2023

The transcription factor interferon regulatory factor 5 (IRF5) is critical for innate immune responses downstream of Toll-like receptors (TLRs). IRF5 is also linked to the pathogenesis of systemic lupus erythematosus (SLE) in humans and mice. However, it remains elusive whether and how IRF5 activity can be negatively regulated. The Src family kinase Lyn is also implicated in human SLE, and Lyn−/− mice develop an SLE-like disease. Here we show that Lyn selectively inhibits IRF5 activity, while it does not affect the NF-κB pathway. Lyn physically interacted with IRF5, thereby suppressing the phosphorylation and ubiquitination of IRF5, post-translational modifications important for IRF5 activation. Interestingly, the kinase activity of Lyn was dispensable for the suppression of IRF5 activity. IRF5 was hyper-activated in TLR7/9-stimulated Lyn−/− bone marrow-derived dendritic cells (BMDCs), and these cells hyper-produced IRF5-dependent cytokines especially type-I interferons (IFNs). IRF5 was constitutively activated (phosphorylated and translocated into the nucleus) in splenic DCs isolated from Lyn−/− mice. Importantly, even monoallelic ablation of the Irf5 gene was sufficient to alleviate the hyper-production of type-I IFNs in TLR7/9-stimulated Lyn−/− BMDCs, and to ameliorate the development of SLE-like symptoms, such as autoantibody production and autoimmune glomerulonephritis, in Lyn−/− mice. Our results identify Lyn as a critical suppressor of the TLR-MyD88-IRF5 pathway, and implicate that the selective control of IRF5 activity may contribute to better therapeutics for SLE.
The role of TWEAK and Fn14 in ultraviolet light-induced cutaneous lupus
Authors: Doerner Jessica L , Chalmers Samantha A. , Friedman Adam , Putterman Chaim
Published: January 2023

Cutaneous lupus erythematosus (CLE) is a common disease manifestation in SLE patients. Ultraviolet-B irradiation (UVB) is well-known trigger of CLE; many lupus patients are photosensitive, and develop skin lesions following sun exposure.

TWEAK, a TNF superfamily member, is a soluble cytokine that binds a sole signaling receptor, Fn14. TWEAK/Fn14 interactions are important in key biologic processes, including angiogenesis, cell death and inflammation, and are often upregulated during tissue injury. We found that MRL-lpr/lpr (MRL/lpr) lupus-prone mice deficient for the Fn14 receptor (Fn14KO) had markedly attenuated skin lesions following UVB irradiation, as compared to Fn14WT mice. Histologically, MRL/lpr Fn14WT mice had more keratinocyte apoptosis, degeneration of the basement membrane, and dermal inflammation. Further evaluation showed decreased infiltration of IBA-1+ macrophages, CD3+ T cells, and NGAL+ cells (neutrophils) in the skin of MRL/lpr Fn14KO mice. Depletion of macrophages in MRL/lpr mice using the CSF1R/fms kinase inhibitor GW2580 attenuated irradiation-induced tissue injury, confirming a pathogenic role for these Fn14-expressing cells in UVB-induced lesions. Several key chemokines (including MCP-1, MIP-1α, MIP-1β) were decreased following macrophage depletion and also differentially expressed between Fn14KO and WT mice, suggesting regulation through TWEAK/Fn14 signaling. Finally, both in vitro and in vivo, Fn14 was upregulated on dead and dying cells following irradiation, which can locally sensitize cells to the effects of TWEAK. Our data reveals important contributors to the pathogenesis of UVB-induced cutaneous lesions, and suggests a novel role for TWEAK/Fn14 signaling in skin disease.