bluuebunny/crossref_metadata_2025 huggingface dataset:
Interferon-β-driven inflammatory B cells promote TH17-mediated neuro-inflammation
Authors: Axtell Robert C. , Agasing Agnieshka , Quinn James L. , Kumar Gaurav , Ko Rose M.
Published: January 2023

Interferon (IFN)-β is a popular therapy for multiple sclerosis (MS). However, neuro-autoimmune diseases that have strong T Helper (TH) 17 signatures are exacerbated with IFN-β; these includes MS patients who are non-responders to IFN-β and patients with neuromyelitis optica (NMO). Previous studies using experimental autoimmune encephalomyelitis (EAE) demonstrated that IFN-β treatment has exacerbated TH17-induced disease. These results are confounding because one proposed mechanism of the efficacy of IFN-β treatment is that it inhibits IL-17 expression in T-cells. Our objective is to elucidate the conflicting theories on how IFN-β affects TH17 function. Here, we demonstrate that IFN-β enhances the effects of IL-23 on TH17 cells and promotes their pathogenicity by elevating the expression of TH1 and TH17 genes as well as GM-CSF. We next determined the precise cellular mechanism by which IFN-β drives an inflammatory TH17 response. We found that the IFN-β exerts its inflammatory effect by directly acting on B cells which in turn effected the function of T helper cells. Specifically, we found that IFN-β elevates the expression of the co-stimulatory receptors CD80 and CD86 as well as the inflammatory cytokine IL-6 in B-cells. Furthermore, we found that B cells previously stimulated with IFN-β drive the differentiation of myelin-specific TH17 cells, which secrete the inflammatory cytokines GM-CSF, IL-17A, IFNγ and IL-6. Taken together, these findings resolve a mechanistic paradox surrounding type 1 interferon and TH17-induced disease and illuminates possible cellular pathways responsible for the pathophysiology of NMO and of MS patients who are IFN-β non-responders.
PD-1 blockade markedly enhances immunity to prostate tumors by adoptively transferred human Vγ2Vδ2 T cells in an NSG mouse model
Authors: Morita Craig T. , Wang Hong , Nada Mohanad H.
Published: January 2023

Human γδ T cells expressing Vγ2Vδ2 TCRs monitor foreign- and self-prenyl pyrophosphate metabolites in isoprenoid biosynthesis to mediate immunity to microbes and tumors. Bisphosphonate treatment of tumors results in increases in isopentenyl pyrophosphate that is sensed by butyrophilin 3A1. This allows Vγ2Vδ2 T cells to recognize and kill all tumor types independent of MHC expression or the number of tumor mutations. In clinical trials, adoptive immunotherapy with Vγ2Vδ2 cells has few side effects but has only resulted in a few partial and complete remissions with the most common response being stable disease. To improve effectiveness, we have now tested the combination of PD-1 blockade with Vγ2Vδ2 T cells and the bisphosphonate, pamidronate. Vγ2Vδ2 T cells were found to express PD-1, CTLA-4, LAG-3, and TIM-3 inhibitory receptors during the 14 day ex vivo expansion. Moreover, tumor expression of PD-L1 was increased by co-culture with activated Vγ2Vδ2 cells and by exogenous IFN-γ. To assess the in vivo effect of PD-1 blockade, immunodeficient NSG mice were inoculated with human PC-3 prostate cancer cells that naturally express PD-L1. After 13 days, the mice were treated with pamidronate followed 1 day later by purified Vγ2Vδ2 cells combined with either an anti-PD-1 or a control IgG1 mAb. This regimen was repeated weekly for 5 weeks. Anti-PD-1 mAb treatment markedly enhanced Vγ2Vδ2 immunity to PC-3 tumors, reducing tumor volume nearly to zero after 5 weeks. These results demonstrate that PD-1 checkpoint blockade can enhance the effectiveness of adoptive immunotherapy with γδ T cells in treating prostate tumors in a preclinical model and suggests that this combination should be tested in patients.
NKT cell ligand-induced cytokines enhance antitumor effects with PD-1 blockade by reinvigorating exhausted CD8 T cells.
Authors: Bae Eun-ah , Seo Hyungseok , Choi Jeongwon , Lee Jeongmi , Kang Chang-Yuil
Published: January 2023

Various immune checkpoint blockades have shown great promise for cancer patients. Anti-PD-1 therapies are known to reinvigorate exhausted immune cells, which progressively have lost effector function after chronic exposure to antigen. Although anti-PD-1 blockades were tested in various clinical trials on a wide range of tumors, many have observed clinical efficacy in only a portion of the patients tested. As such, these anti-PD-1 resistance or non-responsiveness needs to be overcome by combination therapeutic approaches that can synergize with existing immune checkpoint blockades. In this study, we investigated the effects of cytokines induced by invariant Natural Killer T (iNKT) cell ligand stimulation, more specifically α-galactosylceramide (αGC), on exhausted CD8 T cells. We used a mouse tumor model expressing ovalbumin antigen (OVA) and tracked OVA-specific CD8 T cells to show the effects on exhausted CD8 T cells in the tumor. αGC-induced cytokines not only enhanced the effector functions, but also diversified the functionality of exhausted CD8 T cells in vitro. Also, administration of αGC-loaded antigen presenting cells (APCs) restored exhausted CD8 T cell functions and led to higher cytotoxic effects in vivo. Among αGC-induced cytokines, we found IL-2 and IL-12 to be crucial in enhancing exhausted CD8 T cell function in mouse tumors and patient tumors. Furthermore, we showed that αGC-loaded APCs bring synergistic effects with PD-1 blockade in a mouse therapeutic tumor model. Our studies suggest a new insight for applying αGC as an adjuvant in cancer immunotherapy.
Targeting type I interferon tumor entry by anti-PD-L1 creates feedforward antitumor responses to overcome innate and adaptive resistance
Authors: Tang Haidong , Liang Yong , Guo Jingya , Qiu Xiangyan , Peng Hua , Fu Yang-Xin
Published: January 2023

Most patients remain unresponsive to intensive PD-1/PD-L1 blockade therapy despite the presence of tumor-infiltrating lymphocytes. We propose that impaired innate sensing might limit the complete activation of tumor-specific T cells after PD-1 blockade. Local delivery of type I interferon (IFN) restored antigen presentation but also upregulated PD-L1, dampening subsequent T-cell activation. Therefore, we armed anti-PD-L1 antibody with IFN (IFN-anti-PD-L1) to create feedforward loops targeting both PD-L1 blockade and IFN-alpha receptor (IFNAR) activation within tumor microenvironment. A synergistic effect of anti-PD-L1 and IFN was achieved to overcome both PD therapy and IFN resistance with least side effects in advanced tumors. Intriguingly, we observed that PD-L1 in either tumor cells or tumor-associated host cells was sufficient for fusion protein targeting and antitumor effects. IFN-anti-PD-L1 activated IFN receptor signaling in host cells but not tumor cells to initiate T-cell reactivation. Clinically, patients with higher type I IFN levels in tumor tissues show better prognosis after PD-1 blockade therapy. Our data suggest that a next-generation PD-L1 antibody armed with IFN improves tumor targeting and antigen presentation while countering innate or T-cell-driven PD-L1 upregulation and overcoming innate and adaptive tumor resistance to checkpoint blockade therapy.
Single Cell RNA Sequencing of Lung Adenocarcinoma Reveals Heterogeneity of Immune Response-related Genes
Authors: Ma Keyue , Schonnesen Alexandra A. , Brock Amy , Van Den Berg Carla , Eckhardt S. Gail , Liu Zhihua , Jiang Ning
Published: January 2023

Immunotherapies have emerged to be promising approaches to treating cancers, while the partial responses in multiple clinical trials suggest the significance of characterizing intertumoral and intratumoral heterogeneity for better prognosis and decision-making in treatment. The lack of intratumoral characterization of immune response-related genes in cancer cells, however, hinders the further development of metrics to select and predict immunotherapies. Therefore, we analyzed single cell RNA-seq data from lung adenocarcinoma patients to identify intratumoral heterogeneity of immune response-related genes and demonstrated their potential effect on immunotherapy efficacy. We found IFNγ signaling pathway genes are heterogeneously expressed and co-regulated in cancer single cells, including MHC class II molecules (MHCII), which form a set of favorable prognosis markers. The upregulation of MHCII is also mutually exclusive with upregulation of cell cycle pathways that are associated with unfavorable prognosis. Moreover, analysis of two groups of tumor restricted antigens, neoantigens and cancer testis antigens, revealed heterogeneity in their expression in single cells. These analyses provide the rationale of applying combinatorial therapies to prevent tumor escape and basis for future development of prognosis metrics based on intratumoral heterogeneity.
Linkage between TIM3 genotype and human HER2 vaccine response in Diversity Outbred (DO) HER2 transgenic (Tg) mice
Authors: Gibson Heather , Jones Richard , Dyson Greg , Reyes Joyce , Morris John , Wei Wei-Zen
Published: January 2023

Regulatory T cell (Treg) depletion boosts HER2 vaccine response in human HER2 transgenic (Tg) mice. To test whether polymorphisms of Treg-associated genes impact vaccine response, individually distinct F1(DOxB6 HER2 Tg) mice were vaccinated 2× with Adv/HER2 to generate highly variable responses. Single nucleotide polymorphisms (SNP) of 16 candidate genes were analyzed by MonsterPlex sequencing or TaqMan assay, with TIM3 showing an association with vaccine response. There are14 SNPs in the 2kb proximal promoter region and 10 SNPs in a 1kb region containing TIM3 exon 7. Four distinct TIM3 genotypes were identified for either region with one genotype consistently matching between the promoter and exon 7 loci, i.e. the CAST/EiJ strain genotype. Analysis of 84 vaccinated DOxB6 HER2 Tg mice using a tag SNP rs28230613 showed that mice with homozygous CC produce HER2 Ab at 26.9±28.9 μg/mL, and those with heterozygous CA (CAST/Eij genotype) 12.8±19.4 μg/mL (p=0.0075). T cell response was absent in most CA heterozygotes. Relative transcript levels of splenocyte TIM3 over CD4 showed heterozygotes at 2.06±2.27 and homozygotes 0.54±0.51 (p=0.0073, n=24), indicating higher TIM3 transcript in CA heterozygotes. Thus, CAST/EiJ TIM3 genotype is associated with higher TIM3 transcript level and lower vaccine response to self HER2. This correlation is absent in mice depleted of CD25+ Treg, implicating Treg and their TIM3 in regulating HER2 vaccine response. The segregation of TIM3 genotype with vaccine response is not absolute, suggesting additional molecular regulation in HER2 vaccine response. CA76340
CTLA-4 limits anti-CD20-mediated tumor regression
Authors: Ren Zhenhua , Peng Hua , Fu Yang-Xin
Published: January 2023

The inhibition of tumor growth by anti-CD20 antibody (Ab) treatment is mediated by antibody- and complement-dependent cytotoxicity in xenograft tumor models. Additionally, anti-CD20 therapy for B-cell lymphoma can result in intrinsic and extrinsic tumor resistance to further Ab treatment. However, adaptive immune response-related resistance has not been well studied in anti-CD20-mediated tumor control, and adaptive immunity has long been underestimated. However, using a syngeneic mouse B-cell lymphoma model, we observed that CD8+ T cells played an essential role for anti-CD20-mediated tumor regression. Mechanistically, anti-CD20 therapy promoted DC-mediated cross-presentation. Importantly, macrophages were also necessary for the increase in the tumor-specific CTL response after anti-CD20 treatment, via the production of type I IFN to activate DC function. Furthermore, adaptive resistance is gradually developed through the CTLA-4 pathway in Treg cells in larger lymphomas. Further blockade of CTLA-4 can synergize with anti-CD20 treatment in anti-tumor activities. Thus, this study reveals the essential role of adaptive immunity for anti-CD20-mediated tumor regression and opens a new avenue to combine anti-CD20 with immunotherapy to overcome the resistance of advanced B-cell lymphoma in the clinic.
Immunotherapy for melanoma using nonviral plasmid DNA based approach
Authors: Heller Richard , Lundberg Cathryn , Edelblute Chelsea , Arpag-Mcintosh Sezgi , Shi Guilan
Published: January 2023

Malignant melanoma is the deadliest form of skin cancer. New immunotherapy approaches including anti-CTLA4, anti-PD-1 and anti-PD1-L1 have improved this prognosis for several patients. While the results obtained with these therapies are encouraging, there is still a need to establish improved therapies. IL-12 is a potent cytokine mediating antitumor activity. However, it has only achieved modest antitumor effects in clinical trials, often accompanied by unacceptable adverse events. We evaluated if intratumoral delivery of a plasmid encoding IL-12 using gene electrotransfer (GET) could be used to induce an anti-tumor response. Effective protocols delivering plasmids with GET directly into tumors induced not only local immune response, but a systemic one as well. We have been evaluating the specific response following pIL-12 delivery as well as the potential to combine with checkpoint inhibitors to determine if the response could be further augmented. Using a B16F10 mouse melanoma model, we show that melanoma exhibited unique immune cell composition within the tumor microenvironment after intratumoral injection of pIL12 with GET. The total number of memory immune cells was markedly increased in pIL12 GET melanoma groups compared with control group. pIL12 GET displayed significant regulation of multiple immune cell types, including CD8+ cells, regulatory T cells and myeloid cells, which were induced to mount a CD8+ immune response. It was also observed that there was an increase in PD-1 expression following pIl-12 delivery. Taken together, these findings suggest a sequence of immune activity following pIL12 GET. Further, these results suggest that pretreatment or simultaneous treatment with IL-12 may augment anti-PD1 therapy.
The inhibitory NKR-P1B:Clr-b recognition axis facilitates detection of oncogenic transformation and cancer immunosurveillance
Authors: Tanaka Miho , Fine Jason H. , Aguilar Oscar A. , Kirkham Christina L. , Belcheva Antoaneta , Martin Alberto , Ketela Troy , Moffat Jason , Allan David S.J. , Carlyle James R.
Published: January 2023

Natural killer (NK) cells express receptors specific for MHC class I (MHC-I) molecules involved in “missing-self” recognition of cancer and virus-infected cells. Here, we elucidate the role of MHC-I-independent NKR-P1B:Clr-b interactions in the detection of oncogenic transformation by NK cells. Ras oncogene overexpression was found to promote a real-time loss of Clr-b on mouse fibroblasts and leukemia cells, mediated in part via the Raf/MEK/ERK and PI3K pathways. Ras-driven Clr-b downregulation occurred at the level of the Clrb (Clec2d) promoter, nascent Clr-b transcripts, and cell surface Clr-b protein, in turn promoting missing-self recognition via the NKR-P1B inhibitory receptor. Both Ras- and c-Myc-mediated Clr-b loss selectively augmented cytotoxicity of oncogene-transformed leukemia cells by NKR-P1B+ NK cells in vitro and enhanced rejection by WT mice in vivo. Interestingly, genetic ablation of either one (Clr-b+/−) or two Clr-b alleles (Clr-b−/−) enhanced survival of Eμ-cMyc transgenic mice in a primary lymphoma model, despite preferential rejection of Clr-b−/− hematopoietic cells previously observed following adoptive transfer into naïve wild-type mice in vivo. Collectively, these findings suggest that the inhibitory NKR-P1B:Clr-b axis plays a beneficial role in the innate detection of oncogenic transformation via NK cell-mediated cancer immune surveillance, in addition to a pathological role in the immune escape of primary lymphoma cells in Eμ-cMyc mice in vivo. These results provide a model for the human NKR-P1A: LLT1 system in cancer immunosurveillance in lymphoma patients, and suggest it may represent a target for immune checkpoint therapy.
Thymoquinone enhanced the tumoricidal activity of NK Cells against Lung Cancer
Authors: Singh Santosh Kumar , Mishra Manoj K. , Lillard James W. , Singh Rajesh
Published: January 2023

Among different immune cell populations, NK cells are an important component in unleashing the therapeutic activity of immune cells. Therefore, in order to enhance the tumoricidal activity of NK cells, identification of new small-molecule natural products is prominent. In this study, we used a natural compound Thymoquinone (TQ), a constituent of Nigella sativa to enhance the tumoricidal activity of NK cell. We expanded human NK cell (NK-92MI) population derived from human peripheral blood mononuclear cells (PBMCs). Next, expanded NK cells were co-cultured with A549, which is a non-small cell lung cancer cell lung cancer (NSCLC) cell, with or without TQ and after 24h of co-cultured, harvested supernatants were analyzed for IFN-g secretions by ELISA method. This study was further validated using flow cytometry; immunofluorescence, qRT-PCR and western blot techniques. Our result showed TQ increased IFN-γ secretion by NK cells and enhanced NK cell-mediated killing of NSCLC cells. The up-regulation of pro-apoptotic (BAX, BID) and down-regulation of anti-apoptotic (BCL-2, MCL-1) makers confirmed that TQ enhanced the NK cell tumoricidal activity, which is a simple, economic and reliable method to create a new strategy to improve the treatment of NSCLC and has the potential to create a modern class of treatments for advanced lung cancer.
Role of liver-resident innate lymphocytes in tumor progress
Authors: Song Hao , Peng Hui , Sun Rui , Tian Zhigang
Published: January 2023

Over the past decade, the incidence of hepatocellular carcinoma (HCC) is increased dramatically around the world, especially in Asia. There are numerous reasons aggravating the development of HCC. As a cytotoxic cell, the NK cell is vital for controlling cancer development. However, NK cells are heterogeneous with distinct subsets, among which CD49a+NK cells, namely tissue-resident NK cells (trNK), has been barely researched in tumor models. Given the enrichment of trNK cells within the liver, our study aims to investigate the specific role of trNK cell in HCC.

In our study, we used a mouse model of colorectal cancer with liver metastasis, through intrasplenic injection of MC-38 tumor cells. In this model, liver CD49a+ NK cells as well as iNKT cells decreased with the tumor development, whereas DX5+ conventional NK (cNK) cells increased. Furthermore, liver cNK cells upregulated the expression of TIGIT, which is not apparently changed on liver trNK cells. These findings suggested that cNK and trNK cells might play opposite roles in liver tumor growth. Consistent with our hypothesis, we found that liver tumor was greatly restricted in mice devoid of iNKT cells, further confirming that iNKT cells exert a pro-tumorigenic role. With respect to trNK cells, it remains to be further validated whether they really facilitate tumor development. Therefore, based on our findings, we hypothesized that liver-resident innate lymphocytes, including iNKT and trNK cells, can promote the immune escape of tumor cells. However, the underlying mechanism in this progress still needs to be investigated deeply and thoroughly.
Reduced germinal center formation is not sufficient to promote cross-reactive protection against influenza.
Authors: Johnson Jenny Lynn , Keating Rachael , Thomas Paul G. , McGargill Maureen A.
Published: January 2023

Influenza infection continues to be a major health threat with over 9 million cases annually in the United States alone. Vaccination is the best method of preventing infection; however, current vaccines are only 10–60% effective due to a high mutation rate of the virus and difficulty predicting which strains will circulate. Furthermore, current vaccines will have little to no impact on a novel pandemic virus. A universal influenza vaccine would generate an immune response to conserved portions of the virus, allowing for protection against multiple influenza subtypes. We showed that a low dose of rapamycin, given during influenza vaccination, altered the antibody response such that mice were protected against secondary infection with multiple strains of influenza. Rapamycin reduced formation of germinal centers (GC) resulting in a decrease in antibodies specific for the variable region of the vaccine HA. Thus, we hypothesized that rapamycin enhances heterosubtypic protection by reducing generation of high affinity antibodies specific for the variable region of HA, which allows antibodies specific for the conserved regions to be more prevalent. To test this, we investigated whether the reduction in GCs was sufficient to induce cross-reactive antibodies. We depleted GCs during vaccination by removing follicular helper T cells or by transiently blocking CD40L. The reduction of GC reduced the amount of class-switched, influenza-specific antibody, similar to rapamycin-treated mice. However, this was not sufficient to enhance protection following challenge with a virus of a different subtype. These data suggest that while rapamycin reduces germinal center formation, this alone is not sufficient to promote cross-reactive antibodies.
Protective efficacy against live Chlamydia muridarum intravaginal challenge in BALB/c mice immunized with MOMP encapsulated PLGA 85/15 nanoparticles
Authors: Sahu Rajnish , Verma Richa , Dixit Saurabh , Duncan Skyla A. , Singh Shree R. , Dennis Vida A.
Published: January 2023

Chlamydia trachomatis (Ct) remains the leading sexually transmitted bacterial infection worldwide but yet no vaccine to prevent its spread. Attempts to develop vaccines using whole or subunits have failed due to inefficient delivery systems. We have recently developed a Chlamydia nanovaccine by encapsulating its recombinant major outer membrane protein (rMOMP) in poly (lactic acid-co-glycolic acid) (PLGA) 85/15 nanoparticles (PLGA-rMOMP). In the current study, we assessed the systemic and mucosal antibody responses and the protective efficacy of the PLGA-rMOMP nanovaccine against a live C. muridarum (Cm) challenged infection in immunized mice. Female BALB/c mice were immunized subcutaneously three times at two weeks interval with 50 μg each of either PLGA-rMOMP or bare rMOMP and sacrificed two weeks after the last immunization to measure rMOMP-specific systemic and mucosal antibodies. Serum and cervico-vaginal washes were collected to measure IgG including subtypes IgG2a, IgG2b (Th1) and IgG1 (Th2) responses by ELISA. Immunized mice were challenged intravaginally with 105 inclusions forming units (IFUs) of Cm, swabs were collected for three weeks and cultured to quantify bacterial shedding by inoculating McCoy cells. PLGA-rMOMP immunized mice showed enhanced systemic and mucosal IgG antibodies response followed by IgG1 and IgG2b, as compared to bare rMOMP. Moreover, the PLGA-rMOMP immunization induced complete (60% of mice) and partial (40% of mice). Our results show that PLGA-rMOMP stimulates systemic and mucosal antibodies and provides protective efficacy against a Chlamydia infection in mice.
Aberrant innate immune activation facilitates EcoHIV evasion from vaccine-induced protection
Authors: Liu Li , Peng Jie , Tang Hangying , Kwok Hauyee , Nishiura Kenji , Lin Qingqing , Fang Jun , Chen Zhiwei
Published: January 2023

Aberrant innate immune response during acute phase of infection causes persistent viral infection through suppression of T-cell function. Understanding the impact of this aberrant innate immune activation on vaccine-conferred protection is critical regarding development of effective vaccine against chronic disease. Here, using EcoHIV mice model and a novel DC-targeting DNA vaccine that elicited high frequencies of GAG-specific, broadly reactive, and polyfunctional cytotoxic CD8+ T cells, we show that aberrant innate immune activation at early stage of infection rendered EcoHIV resistant to vaccine protection by suppressing T cells proliferation and function. We found that despite high frequencies of p24-specific IFN-γ+CD8+ T cells elicited by vaccination, EcoHIV have fallen short of effective recall of antiviral T cell immunity induced by prophylactic vaccination in vivo. Provirus DNA was readily detected in PBMC and spleen in all of the infected mice without detectable resistant mutations. Compared to non-immunized mice, provirus copies in immunized mice were decreased only at 1 and 2 weeks post viral inoculation without further decrease over the following weeks. Further investigation revealed that EcoHIV infected multiple DC subsets, and CD11b+ myeloid derived cells within 7 dpi, and induced quick expansion of eosinophils and monocytic cells in spleen, which peaked at 14 dpi and remained high at 21 dpi. The expansion of monocytic cells was associated with down-regulation of CD80 and CD86, up-regulation of PD-L1/L2 on multiple dendritic cell subsets, PD-1+ and Tim+ CD8 T cells, compromised capacity of DCs in stimulating CD4 and CD8 T cells proliferation, and suppressed viral-specific CD8 T cells proliferation in vitro.
CD8+ T cell-mediated inhibition of HIV-1.
Authors: Xu Yinyan , Abad Maria , Warren Joanna A. , Clutton Genevieve , Goonetilleke Nilu
Published: January 2023

CD8+ T cells contribute to the control of viral infections, including HIV-1. Previous studies have reported that CD8+-mediated virus inhibition is predictive of subsequent HIV plasma viremia and CD4+T cell decline in HIV-infected, antiretroviral naïve individuals; higher virus inhibition is associated with a better clinical outcomes.

Ex vivo CD8+ T cell-mediated inhibition of HIV replication is measured after co-culture of CD8+ T cells with HIV-1 super-infected, autologous CD4+ T cells, at different effector to target ratios. We adapted and standardized the CD8+ T cell virus inhibition assay (CD8-VIA) for use as an endpoint assay in therapeutic testing of HIV-1 T cell vaccines.

CD8+ T cells from HIV infected, durably (>2 years) antiretroviral suppressed participants (n=11) were co-cultured with JR-CSF infected autologous CD4+ T cells at E:T ratios ranging from 1/2:1 to 1/160:1, generating CD8+ T cell virus inhibition curves for each participant. IC50 values varied by 100-fold across participants suggesting that CD8+ T cells harbor a broad range of virus inhibitory capacity even when viremia in participants is durably suppressed.

Ongoing studies are comparing IC50 values to other measurements (magnitude, specificity, avidity, proliferation and production of cytokines and lytic molecules) of HIV-specific T cell immunity to understand the critical determinants of CD8+ T cell inhibitory capacity.
A viral homologue of IPS-1 Interacts with TRAF3 leading to a diminished antiviral state.
Authors: He Buyuan , Sanchez Julio C. , Sanchez David Jesse
Published: January 2023

Kaposi’s Sarcoma (KS) is an angioproliferative disease with symptoms including skin lesions that appear at different sites of the body caused by Kaposi’s Sarcoma Associated Herpesvirus (KSHV). The KSHV genome encodes genes pirated from the human genome that are homologous to cellular proteins that disrupt innate immune pathways. We have found a novel ORF in the KSHV genome that is homologous to the human IPS-1 protein. This viral IPS-1 proteins (vIPS-1) contains the PRO domain of IPS-1 but lacks the CARD and TM domains. We hypothesize that vIPS-1 is able to interact with the cellular TRAF3 protein. Western blot data shows that co-expression of vIPS-1 does not affect RIG-I, decreases the stability of human IPS-1 and TRIF, and increases the stability of TRAF3. Also, vIPS-1 interacts with TRAF3 via coimmunoprecipitation, and by co-expressing vIPS-1 with different TRAF3 mutants we found that vIPS-1 interacts with TRAF3 through the TRAF domain which is specifically required for TRAF3-mediated type I IFN production. Furthermore, our data shows that while expression of IPS-1 is able to inhibit VSV-GFP replication, expression of vIPS-1 significantly enhances the VSV-GFP replication, consistent with our hypothesis that vIPS-1 blocks the cellular immune responses. In conclusion, the KSHV vIPS-1 protein blocks antiviral immunity by interacting with TRAF3 leading to a decrease in antiviral activity.
Discrepancy between neurokinin 1 receptor antagonist treatment and neurokinin 1 receptor knockout mice in the CD8 T cell response to corneal HSV-1 infection
Authors: Jerome Andrew , Suvas Susmit
Published: January 2023

The purpose of this study is to evaluate the effect blocking neurokinin 1 receptor (NK1R) signaling has on the CD8 T cell response to corneal herpes simplex 1 (HSV-1) infection, using NK1R antagonist (L 760735) or NK1R−/− mice. Corneal HSV-1 infection was carried out on C57BL/6 and NK1R−/− mice. Our results showed an enhanced CD8 T cell response to corneal HSV-1 infection by NK1R−/− mice compared to C57BL/6 mice. The data illustrates that at day 5 post infection, draining lymph nodes (DLNs) in NK1R−/− mice exhibit an increased CD8 T cell IFN-γ response as measured by intracellular cytokine staining. In addition, an increased CD8 T cell proliferation is noted in NK1R−/− mouse DLNs. Interestingly, L 760735 treatment given to HSV-1 infected C57BL/6 mice did not show an increase in CD8 T cell proliferation or IFN-γ production; in comparison to infected, vehicle treated control mice. To determine the underlying cause of this discrepancy, we detected pre-existing LYVE+ vessels in unmanipulated corneas of NK1R−/− mice. This was associated with higher levels of VEGF-C protein in ocular tissue (cornea + limbal conjunctiva) of unmanipulated NK1R−/− compared to C57BL/6 mice, as measured via western blot. We recently reported a massive accumulation of CD11b and CD11c expressing cells near the limbal area in unmanipulated eyes of NK1R−/− mice [J Immunol., 2016, 197 (10) 4021–4033]. Confocal microscopy results showed VEGF-C colocalization with these cells near the limbal area in NK1R−/− mice. Since corneal lymphatics play an important role in generating the CD8 T cell response to corneal HSV-1 infection, our results suggest an enhanced CD8 T cell response in NK1R−/− mice, likely due to a notable increase in corneal lymphatics in NK1R−/− compared to C57BL/6 mice.
Female sex hormones regulate the IL-36 family members in the female reproductive tract and impact HSV-2 pathogenesis
Authors: Gardner Jameson K. , Alexander Thessaly , Herbst-Kralovetz Melissa M.
Published: January 2023

Recently, the contraceptive Depo-Provera (DMPA) has been linked to an increased risk of sexually transmitted infection acquisition (e.g. HSV-2 and HIV). However, a fundamental gap exists in the understanding of specific mechanisms whereby DMPA use increases risk for HSV-2 infection. IL-36γ is a novel regulator of mucosal inflammation in the female reproductive tract (FRT) and we have shown that IL-36γ is induced in response to HSV-2. One study reported that IL-36γ was the only cytokine upregulated in cervicovaginal secretions from HSV-2 seropositive DMPA users, suggesting that IL-36γ may be a link between DMPA use and HSV-2 infection. We hypothesize that DMPA suppresses IL-36γ expression in the vaginal environment, thereby increasing susceptibility to genital HSV-2 infection. We found that DMPA treatment in mice decreased expression of the IL-36 cytokines in the FRT, while β-estradiol treatment increased expression. To evaluate the impact of IL-36γ on HSV-2 pathogenesis, we used an intravaginal challenge model and treated mice with DMPA to enhance susceptibility to infection. Mice treated with IL-36γ 4h prior to lethal challenge exhibited a delay in disease onset, less severe disease, decreased vaginal viral replication, increased disease resolution and overall survival. IL-36γ treatment increased levels of pro-inflammatory cytokines in vaginal lavages 4h after treatment, and neutrophil recruitment was enhanced and corresponded with increased levels of chemokines. Together, we demonstrate that IL-36γ is hormonally regulated in the FRT and that treatment with IL-36γ promotes the transient induction of immune mediators and neutrophil recruitment in the vaginal microenvironment that result in increased HSV-2 clearance.
Ectromelia virus suppresses cathepsins and cystatins expression at both mRNA and protein levels in dendritic cells
Authors: Bossowska-Nowicka Magdalena , Mielcarska Matylda Barbara , Kaczmarek Monika M. , Romaniewicz Marta , Struzik Justyna , Toka Felix Ngosa , Szulc-Dabrowska Lidia
Published: January 2023

Cathepsins belong to cysteine proteases and play essential roles in processing of internalized peptides during antigen presentation in the context of the major histocompatibility complex (MHC) class II molecules. Function of cathepsins is regulated by cystatins – endogenous protein inhibitors. Viruses, such as poxviruses, have evolved many mechanisms to escape immune responses after infection. Our studies are focused on ectromelia virus (ECTV, a poxvirus closely related to variola virus, VARV – a causative agent of smallpox), and its influence on cathepsins or cystatins. Our results show that ECTV down-regulates gene and protein expression of selected cathepsins and cystatins in infected murine JAWS II dendritic cells (DCs) and GM-CSF-derived bone marrow cells (GM-BM), composed of conventional DCs and macrophages. Moreover, the ability to endocytose and process a soluble antigen is reduced in JAWS II and GM-BM cells during ECTV infection. After knockdown of cathepsins and cystatins using siRNA in JAWS II DCs, the virus titer increases when compared with control cells. The inhibition of cathepsins and cystatins together with elevated virus titers during the absence of these proteases may be a viral strategy to escape immune responses and simultaneously enable the virus to replicate effectively in infected cells. Importantly, defining the poxvirus-host interactions, including lysosomal proteases, may lead to development of potential therapeutic targets or vaccination strategies.
Effect of interferon-λ1 on the secretion of IP-10 by hepatitis C virus-infected cells and the migration of natural killer cells
Authors: Yoon Joo Chun , Lee Woojeong
Published: January 2023

Hepatitis C virus (HCV) is an important human pathogen because it causes chronic hepatitis, liver cirrhosis, and hepatoma. Interferon (IFN)-λ1, which inhibits viral replication, and natural killer (NK) cells, which kill virus-infected cells, would be important in the innate immunity to HCV infection. Although it cannot directly stimulate NK cells because of the absence of its receptors on NK cells, IFN-λ1 is able to bind the receptors on HCV-infected hepatocytes. This study investigated, therefore, whether the stimulation of HCV-infected hepatocytes by IFN-λ1 treatment modulated NK cell function. Cell-culture-generated HCV virions, hepatoma cell lines, such as Huh7.5 and HCV-core replicon Huh7 cells, and primary human NK cells were employed. IFN-λ1 responses and NK cell function were measured by qPCR, ELISA, western blot, migration assay, and flow cytometry. IFN-λ1 effectively reduced HCV replication and increased the expression of interferon-stimulated genes (ISGs), such as MX1 and IFIT1, in HCV-infected cells and replicon cells. In addition, IFN-λ1 enhanced the transcription of chemokines such as IP-10 and IL-8. In particular, IP-10 secretion by HCV-infected Huh7.5 cells and HCV-core replicon Huh7 cells was significantly increased after IFN-λ1 treatment. Although the concentration of IP-10 secreted by HCV-infected Huh7.5 cells in vitro was insufficient to elicit considerable migration of NK cells, recombinant IP-10 could induce migration of NK cells ex vivo. In conclusion, IFN-λ1 enhanced IP-10 secretion by HCV-infected cells and IP-10 could modulate NK cell migration. These results suggested that IFN-λ1 would indirectly regulate the chemotaxis of NK cells through augmenting IP-10 secretion by HCV-infected cells.
Interleukin-36 cytokines regulate antiviral immunity in the skin and keratinocytes
Authors: Jensen Liselotte E. , Wang Peng
Published: January 2023

Pathogens develop immune evasion strategies that delay host immune responses. How the host has adapted counter strategies is often less clear. Herpes simplex virus (HSV) blocks inflammatory signaling from infected cells by preventing extracellular release of IL-1β. Previously, we reported that HSV infected keratinocytes retain the ability to signal danger to the surrounding tissue through secretion of IL-1α. This inflammatory signal appeared essential in vivo for early recruitment of immune cells to infected sites. Furthermore, mice deficient of the common receptor for IL-1α and IL-1β, IL-1R1, were more susceptible to lethal outcome of HSV infection. Hence, IL-1α may have evolved to counteract the HSV immune evasion mechanism targeting IL-1β. Here, the IL-1 related IL-36α, IL-36β and IL-36γ are studied. Like the IL-1 cytokines, the IL-36 proteins lack signal peptides for conventional protein secretion. The IL-36α mRNA was the principal IL-36 induced in keratinocytes by HSV-1 in vitro. Surprisingly, using the flank skin HSV-1 infection model, IL-36α knockout (KO) mice developed disease indistinguishable from that observed in wild type mice. In contrast, IL-36β KO mice progressed to more severe disease, whereas IL-36γ KO mice only developed modestly larger skin lesions. This could suggest that HSV-1 promotes retention of IL-36α in a manner resembling that restricting IL-1β activity. IL-36β on the other hand may achieve the highest degree of activity through a yet to be determined mechanism. Viral loads were elevated in skin from IL-36β KO mice. Furthermore, pre-treatment of human keratinocytes delayed progression of HSV-1 infections. These observations suggest that IL-36β regulates innate antiviral mechanisms in keratinocytes.
Spontaneous translocation of a human enterococcal gut pathobiont drives systemic autoimmunity
Authors: Vieira Silvio Manfredo , Hiltensperger Michael , Kumar Varun , Zegarra-Ruiz Daniel , Dehner Carina Anja , Barbieri Andrea , Jain Dhanpat , Goodman Andrew , Kriegel Martin A.
Published: January 2023

Host-microbiota interactions in the pathogenesis of autoimmunity remain poorly understood. Here, we show that a gut commensal, Enterococcus gallinarum, reaches lymphocytes beyond the gut barrier in the mesenteric lymph node, liver and spleen of lupus-prone (NZWxBXSB)F1 mice. Oral vancomycin treatment suppressed growth of E. gallinarum in tissues of these mice, prevented organ manifestations and mortality by lowering pathogenic autoantibodies, Th17 and Tfh cells. Hepatocyte-commensal cocultures revealed induction of the lupus signature cytokine IFN-α as well as the AhR pathway (AhR, Cyp1a1, Cyp1a2) that could be linked to Th17 induction in vivo. E. gallinarum monocolonization in germ-free C57BL/6 animals allowed for translocation to internal organs and induction of autoantibodies as well as increase of lamina propria plasmacytoid dendritic cells (pDCs) and systemic Th17 cells. Additionally, RNA-Seq of short-term ileum E. gallinarum-monocolonized mice demonstrated induction of molecules related to barrier function (occludin, claudins, Plvap, Axin2), the mucus layer (mucin, Fut2), antimicrobial defence (Reg3b, Defa2), and inflammation and pDC induction (AhR/Cyp1a1, Enpp3). Species-specific PCR of sterilely obtained liver tissue from autoimmune hepatitis and lupus patients revealed also E. gallinarum, suggesting similar processes in humans. Accordingly, human hepatocyte-commensal coculture demonstrated production of the same autoimmune-promoting factors as with murine hepatocytes. Collectively, these data indicate that a human pathobiont translocates spontaneously to promote autoimmunity in genetically predisposed hosts, broadening our understanding of autoimmune host-microbiota interactions.
Gut commensal-dependent production of autoantibodies against the primordial lupus autoantigen Ro60 in the absence of mammalian Ro60.
Authors: Datta Rahul , Dehner Carina Anja , Ruff William Edwards , Greiling Teri , Kriegel Martin A.
Published: January 2023

Introduction

The RNA-binding protein Ro60 is a primordial autoantigen in systemic lupus erythematosus (SLE), which is highly conserved across multiple species. We have previously linked anti-human Ro60 T cell and antibody responses to human commensals carrying Ro60 orthologs. Here, we tested in vivo if these autoantibodies can arise due to the murine gut microbiota in the absence of host Ro60 protein.

Methods

Lupus-prone TLR7 tg B6 mice were crossed to Ro60 KO B6 mice and followed longitudinally for Ro60 IgG ELISAs. Littermates were treated with broad-spectrum antibiotics (vancomycin, neomycin, ampicillin, metronidazole) to determine if depletion of the gut microbiota affects anti-Ro60 autoantibody induction and disease manifestations. High-throughput 16S rRNA sequencing of the V4 region was performed on the MiSeq platform.

Results

TLR7 tg mice produced persistently anti-Ro60 autoantibodies despite crossing to Ro60 KO mice. Oral administration of antibiotics significantly lowered anti-Ro60 autoantibodies (n=6 each, p=0.007). 16S sequencing of antibiotics-fed mice demonstrated outgrowth of Streptophyta but suppression of taxa that may contain commensal Ro60 orthologs (Clostridiaceae, Bacteroides).

Conclusions

Mice genetically prone to SLE-like disease can mount autoimmune responses against Ro60 even in the absence of the autoantigen. These findings suggest an antigenic trigger within the microbiota, which is supported by interventional studies with antibiotics and 16S sequencing. In summary, these studies suggest a mechanism for the development of lupus which extends beyond the known traditional risk factors.
OX40 signaling Induces Canonical Antigen Presentation-independent Proliferation of Human and Murine Thymic Regulatory T-cells
Authors: Kumar Prabhakaran , Marinelarena Alejandra , Bhattacharya Palash , Epstein Alan L. , Prabhakar Bellur S.
Published: January 2023

Regulatory T-cells (Tregs) play a pivotal role in immune-tolerance, and loss of their function can lead to autoimmunity. Natural Tregs generated in the thymus contribute substantially to the Treg pool in the periphery where they suppress self-reactive Teff responses. Recently, we showed that OX40L (TNFSF4) can drive selective proliferation of peripheral Tregs independent of canonical antigen presentation (CAP-independent) in the presence of low dose IL-2. Therefore, we reasoned that OX40 signaling might be integral to the TCR-independent phase of murine and human thymic Treg (tTreg) development. Thymic Treg development is a two-step process: Strong T-cell receptor (TCR) signals combined with co-signals from the TNF receptor Super Family (TNFRSF) members facilitate tTreg precursor selection, followed by a TCR-independent phase of tTreg development when their maturation is driven by IL-2. Therefore, we investigated whether OX40 signaling can also play a critical role in the TCR-independent phase of tTreg development. OX40−/− mice had significantly reduced numbers of CD25−Foxp3low tTreg precursors and CD25+Foxp3+ matured tTregs, while OX40L treatment of WT mice induced significant proliferation of these cell subsets. Relative to tTeffs cells, OX40 was expressed at higher levels in both murine and human tTreg precursors and matured tTregs. In ex vivo cultures, OX40L increased tTreg maturation and induced CAP-independent proliferation of both murine and human tTregs which was mediated through the activation of AKT-mTOR signaling. These novel findings show an evolutionarily conserved role for OX40 signaling in tTreg development.
Ocular autoimmunity develops despite concurrent absence of Th1 and Th17 signature cytokines
Authors: Bing So Jin , Silver Phyllis B. , Jittayasothorn Yingyos , Horai Reiko , Caspi Rachel R.
Published: January 2023

Th1 and Th17 cells are each a standalone effector phenotype, and each can elicit ocular autoimmunity independently of the other. In this study, we used mice deficient in IL-17A, or IFN-gamma (GKO), or both (DKO) to dissect the role of Th1 and Th17 cytokines in experimental autoimmune uveitis (EAU). EAU was induced by active immunization with the retinal autoantigen IRBP. Previous studies showed that EAU in GKO mice was exacerbated, whereas it was attenuated in IL-17A KO mice. Surprisingly, DKO mice were fully susceptible to EAU with scores similar to wild type (WT) controls, and displayed eosinophil-dominant infiltration along with enhancement of IL-17F, Th2, and proinflammatory cytokines. To test which cytokines contributed to pathology, EAU-challenged DKO mice were treated with blocking antibodies to IL-17F, TNF-a, GM-CSF or IL-4Ra (a shared subunit of receptor for IL-4 and IL-13). Blockade of IL-17F or TNF-a in DKO mice did not affect the severity of EAU. Treatment with an anti-GM-CSF antibody during both induction and expression stages significantly suppressed disease severity in DKO, but not in WT mice. By contrast, treatment of DKO mice with an anti-IL-4Ra antibody worsened EAU. Importantly, a single intravitreal injection of an anti-GM-CSF antibody after the disease onset reduced EAU severity in both WT and DKO mice. These results suggest that, in the absence of both IFN-gamma and IL-17A, (a) GM-CSF plays a major and nonredundant role as a pathogenic cytokine, (b) the Th2 cytokines IL-4 and/or IL-13 are protective, and (c) IL-17F and TNF-a may be dispensable. Our results highlight that ‘classical’ Th1 and Th17 cells are not the only pathogenic effectors, and that other inflammatory cytokine(s) can contribute to uveitis.
Islet-infiltrating effector and regulatory T cells specific for a single epitope possess distinct, but overlapping TCR repertoires.
Authors: Jing Yi , Kong Yuelin , Bettini Matthew L. , Bettini Maria
Published: January 2023

Regulatory T cells (Tregs) have been shown to play a critical protective role in type 1 diabetes. However, TCR specificity of islet-infiltrating Tregs and its influence on T cell function during autoimmune diabetes is largely unknown. It has been shown that Tregs have a higher affinity for self and possess a unique TCR repertoire. Accordingly, we observed increased TCR signaling in islet-infiltrating insulin-tetramer labeled Tregs compared to effector T cells (Teffs), as measured by the Nur77-GFP reporter of TCR activation. In order to obtain the profile of Treg and Teff TCR repertoires specific for a single epitope, we isolated and sequenced insulin tetramer binding Teffs and Tregs from islets of NOD mice expressing a fixed alpha chain of an insulin specific TCR. Surprisingly, TCR sequence analysis showed relatively high similarity (Morisita-Horn index: 0.27) between Treg and Teff TCR repertoires, where a substantial portion (46.4%) of Tregs expressed shared TCRs. Collectively, our data suggest that Treg and Teff specific for a single epitope can express the same TCRs, which they exploit to exert different effector or suppressive functions.
Cell-specific metabolic models reveal novel metabolic regulators of Th17 pathogenicity: from single-cell RNA-Seq to actionable metabolic targets
Authors: Wagner Allon , Wang Chao , DeTomaso David , Koul Arman , Regev Aviv , Kuchroo Vijay K. , Yosef Nir
Published: January 2023

Cellular metabolism is a powerful regulator of immune response. In order to unbiasedly search for novel metabolic regulators of Th17 development and function, we have developed COMPASS, a computational algorithm to characterize the metabolic landscape of single cells based on single-cell RNA-Seq profiles and flux balance analysis. We used COMPASS to characterize the metabolic heterogeneity in Th17 cells, whose pathogenic state triggers auto-immunity, yet whose non-pathogenic form promotes tissue homeostasis and barrier functions. COMPASS recovered known metabolic switches and predicted that the polyamine pathway should be a novel, powerful regulator of Th17 pathogenicity. We validated the pathway’s effect through an array of transcriptome, LC/MS metabolome, and functional assays. Deletion of polyamine enzymes in T cells resulted in altered metabolic space, T cell functions and, most importantly, aggravated symptoms in EAE, a murine model of multiple sclerosis. While our study is concerned with Th17 cells, COMPASS is generally applicable, and can be used to unbiasedly characterize the metabolic states of any cell population based on its single-cell transcriptome profiles. Furthermore, COMPASS predicts metabolic switches between cell states that present testable, mechanistic hypotheses.