bluuebunny/crossref_metadata_2025 huggingface dataset:
PP4 inhibition stimulates anti-tumor immunity in ovarian cancer
Authors: Curtis Marion R , Raja Remya , Wu Christopher , Rubino Thomas , Utagawa Emma , Magtibay Paul , Butler Kristina
Published: January 2023

Background

Increased infiltration of T cells into ovarian tumors has been repeatedly shown to be predictive of enhanced patient survival. However, despite the evidence of an active immune response in OC, immune checkpoint blockade therapy has been ineffective. Recently studies have shown that deficiencies in the DNA damage response (DDR) can drive increased genomic instability and tumor immunogenicity. One target involved in the DDR that has the potential to be of therapeutic value is protein phosphatase 4 (PP4); however, the effect of PP4 deficiency on anti-tumor immunity remain unknown.

Results

Our results show that PP4 inhibition combined with carboplatin leads to increased carboplatin sensitivity, DNA damage, and micronuclei formation. Using a panel of ovarian cancer cells, we show that PP4 inhibition triggers inflammatory signaling via NFκB and STAT1 activation resulting in increased expression of pro-inflammatory cytokines including IFNβ1, CCL5, CXCL10, and IL-6. We further show that PP4 inhibition and carboplatin-induced STAT1 activation is significantly blunted upon STING knockdown in ovarian cancer cells. Moreover, using mouse ID8 ovarian cancer cell line and OT-I cells, we demonstrate that the combination of PP4 inhibition and carboplatin significantly increased CD8 T cell migration over carboplatin treatment alone. In addition, the same treatment resulted in enhanced NK cell-mediated tumor cell killing and T cell killing in an autologous human organoid model.

Conclusions

Our work has identified a role for PP4 inhibition in promoting anti-tumor immune activation. These findings provide the rationale for combining PP4 inhibitors with immunotherapy as a new approach in ovarian cancer treatment.

Supported by DOD W81XWH2110489
Complement C3 deficiency enhances anti-CD47 efficacy in murine ovarian cancer model.
Authors: Suzuki Sora , Giridharan Thejaswini , Khan A N M Nazmul , Emmons Tiffany R , Yaffe Michael B , Weiskopf Kipp , Das Madhumita , Eng Kevin H , Zsiros Emese , Segal Brahm H
Published: January 2023

Epithelial ovarian cancer (OC) is the most lethal gynecological malignancy. While OC is an immunogenic tumor, single-agent anti-PD-1 have limited efficacy in recurrent OC. Blockade of the CD47/SIRPα pathway, a phagocytosis checkpoint in macrophages and other myeloid cells, can enhance myeloid-driven antitumor immunity and tumor control. However, the benefit of targeting CD47/SIRPα in recurrent OC has been limited. Prior work from our lab and others point to complement priming of innate immune responses as a barrier to T-cell-driven anti-tumor immunity. However, in the specific context of anti-CD47 therapy, complement opsonization of tumor cells could augment the capacity of myeloid cells to phagocytose or injure tumor cells. The role of complement deficiency with anti-CD47 therapy in tumor control is a gap in the field, we therefore evaluated the efficacy of anti-CD47 in female wild-type (WT) versus C3−/− and C5aR1−/− with syngeneic OC (ID8). Mice (n=8–10 per group) were administered IP ID8 tumor cells followed by anti-CD47 or isotype twice weekly for 3 weeks and were monitored for the ascites development and survival. On day 84, all WT mice treated with isotype had ascites by visual inspection and 3 of 10 died. The proportion of mice with ascites varied in other groups, C3−/− mice treated with anti-CD47 having the lowest proportion, 4 of 9 mice with ascites and no deaths (Fisher’s test, WT-isotype vs C3−/− + anti-CD47, p= 0.01). A limitation is the potential endogenous complement production by tumor cells, which will be evaluated. These results suggest a potential benefit for dual targeting of the CD47/SIRPα and complement pathways in OC. Additional studies will probe immunologic correlates and survival benefits of these therapies in OC.

This work was supported by Roswell Park Cancer Center Support Grant P30CA016056 and Apellis Pharmaceuticals, Inc
Malignant cell expression of the aryl hydrocarbon receptor induces PD-L1 and immunosuppression in models of oral and lung cancer
Authors: Snyder Megan , Kenison Jessica , Lara Brian , Wang Zhongyan , Pichardo Tachira , Yang Kangkang , Mazzilli Sarah , Sherr David
Published: January 2023

Immunotherapy has shown dramatic results in treating cancer but only in a minority of patients, so there is unmet need to understand the regulation of immune suppression in tumors. We have shown that the aryl hydrocarbon receptor (AhR) is a central player in regulating immune checkpoints in oral squamous cell carcinoma (OSCC) and may play a similar role in lung cancer. Orthotopic transplant of mouse AhR-knockout (KO) OSCC (MOC1) cells led to complete rejection of tumor formation coupled with increased T cell activation in tumor draining lymph nodes (tdLNs) and T cell signaling in tongues, whereas wild type (WT) OSCC challenge led to tumor formation and upregulation of T cell exhaustion pathways and checkpoint molecules. Similar patterns of AhR-facilitated immune protection appear in our lung cancer model. On average, mice transplanted subcutaneously with CMT167AhRKO lung adenocarcinoma cells survive up to twice as long as their WT-challenged counterparts; up to 50% of CMT167AhRKO-transplanted mice never grow tumor and also were partially protected from challenge with CMT167WT cells. CMT167WT-transplanted mice exhibited increased numbers of CD4+ and CD8+ in tdLNs and decreased T cell trafficking to tumors while the proportion of PD1+, CTLA4+ and Lag3+ CD4+ and CD8+ T cells were significantly lower in CMT167AhRKO tumors. AhRKO in both cell lines decreases PD-L1 expression by 50%, suggesting a mechanism of immune escape by WT tumors. Thus, the immunological protection following transplantation of both AhRKO CMT167 and MOC1 cells suggests downregulation of PD-L1 on malignant cells is at least partially responsible for immune enhancement in AhRKO cells and supports AhR’s potential as a viable immunotherapeutic target in at least two cancers.

Supported by grants from the NIH (R21 ES029624-01, R01 ES029136) as well as Find the Cause Breast Cancer Foundation and Johnson & Johnson Lung Cancer Initiative.
Immunological profiling of tumor-infiltrating CD8+ T lymphocytes in non-small cell lung cancer, head and neck squamous cell carcinoma, breast cancer, and renal cell cancer
Authors: Miller William L , Koguchi Yoshinobu , Kaufmann Johanna K , Yanamandra Niranjan , Griffin Sue , Smothers James , Redmond William L
Published: January 2023

To explore whether the variation in clinical response to immune checkpoint blockade (ICB) reflects intrinsic characteristics of tumor-infiltrating lymphocytes (TIL), TILs from multiple tumor types were analyzed by multiparameter flow cytometry. Recent work identified CD39+ CD103+ double positive (DP) CD8 TIL as tumor-reactive, therefore we assessed this phenotype in primary NSCLC (n=28), BCa (n=23), HNSCC (n=23), and RCC (n=23) specimens. TILs from NSCLC and HNSCC had significantly higher frequencies of DP T cells than BCa and RCC (NSCLC: median=34.2, IQR=15.0–53.8%; HNSCC: median=28.6%, IQR=13.5–44.5%), while TILs from BCa and RCC had low frequencies of DP T cells (BCa: median=2.4%, IQR=1.1–3.9%; RCC: median=4.2%, IQR=1.5–16.6%). Additionally, DP cells in NSCLC and HNSCC were co-expressed immune checkpoint markers PD-1, TIM-3, and LAG-3 at a higher frequency than RCC or BCa (P<0.05). DP CD8 T cells in NSCLC and HNSCC also exhibited increased effector potential (granzyme B+) as compared to BCa and RCC, suggesting functional differences. Expression of the transcription factor Eomesodermin, associated with T cell exhaustion, was higher in effector CD8 T cells from RCC (median=84.5%, IQR=44.9–97.4%) as compared to NSCLC (median=28.7%, IQR=20.7–61.4%), BCa (median=30.1%, IQR=0–50%), and HNSCC (median=40.3%, IQR=20.1–61.2%). Additionally, 4-1BB, an indicator of TCR engagement, trended higher in RCC (mean=8.6%, IQR=1.4–46.7%) as compared to BCa (median=1.9%, IQR=0.4–8.6%). These data highlight the heterogeneity of human TILs isolated from distinct tumor types and provide insight into the basal expression of actionable therapeutic targets.

Supported by GlaxoSmithKline and the Providence Portland Medical Foundation
Identification of a subset of tumor-reactive CD8 TILs expressing NKG2A in HNSCC and CRC
Authors: Fesneau Olivier , Samson Kimberly , Rajamanickam Venkatesh , Ross David , Duhen Thomas
Published: January 2023

Even though blocking the interaction of NKG2A with its ligand HLA-E is a promising strategy to restore the function of cytotoxic T cells and induce tumor cell killing, several aspects of the biology of NKG2A+ CD8 T cells remain to be understood to fully benefit from this therapeutic approach. Here, we characterize the expression of NKG2A on tumor-infiltrating CD8 T cells (CD8 TILs) in patients with head and neck squamous cell carcinoma (HNSCC) and microsatellite-stable colorectal cancer (CRC). Our results show that NKG2A is primarily expressed by a population of tumor-reactive CD8 TILs identified by coexpression of CD39 and CD103 (DP CD8). NKG2A expression was stable and maintained during in vitro expansion. Interestingly, while TCR stimulation in presence of IL-15 and TGF-β are thought to be responsible for NKG2A upregulation, our in vitro results suggest that other factors might be necessary to induce its expression on naive CD8 T cells. Ex vivo analysis of NKG2A+ DP CD8 TILs by scRNAseq and flow cytometry revealed that those cells were activated, more differentiated, and displayed a cytolytic profile suggesting a role in tumor recognition and killing. Finally, we found that, even though there was a high degree of clonal overlap between NKG2A+ and NKG2A− DP CD8, some CDR3 TCR sequences were enriched in one versus the other subset. Consequently, HPV-reactive cells were preferentially enriched in NKG2A− DP CD8 TILs. Altogether, our results provide evidence that NKG2A is expressed by tumor-reactive CD8 TILs in HNSCC and CRC. However, NKG2A-inducing factor(s) remain to be identified and might provide new potential drug targets to bolster anti-tumor immune responses in cancer patients.

Supported by the Providence Portland Medical Foundation.
Multimodal single-cell analysis of human TILs across multiple tumor types reveals heterogeneity and potential opportunities for personalized immunotherapy
Authors: Redmond William L , Koguchi Yoshinobu , Miller William L , Christie Tanisha , Kaufmann Johanna , Seestaller-Wehr Laura , Yanamandra Niranjan , Griffin Sue , Smothers James
Published: January 2023

Immune checkpoint blockade (ICB) efficacy varies among tumor types likely due to differences in tumor infiltrating lymphocyte (TIL) composition and function within the tumor microenvironment (TME). To help understand these differences, we conducted multimodal single-cell analysis of TILs including single-cell RNA sequencing (scRNA-seq), CITE-seq (oligo-tagged antibodies), and scTCR-seq (10× Genomics) in (non-small cell lung cancer: NSCLC; head and neck squamous cell carcinoma: HNSCC; renal cell carcinoma: RCC; and breast cancer: BrCa; n=48). We found that regulatory T cell (Treg) frequency was higher in HNSCC, whereas exhausted T cells (Tex) were higher in NSCLC and RCC. In contrast to other tumor types, Tex in RCC lacked the expression of CD103, a hallmark of tissue-resident T cells. On the other hand, expression of PD-1, TIM-3, and LAG-3 were more prominent in Tex in RCC. Interestingly, Tex in HNSCC showed higher expression of TIGIT than other tumor types. Previous work has demonstrated an increased presence of CD4+CD8+ double-positive T cells (DPT) in RCC, which was associated with better overall survival. Therefore, we used CITE-seq to identify DPT and then compared the composition of DPT among different tumor types. DPT were CD39+, a marker for tumor-reactive T cells, and the vast majority were transcriptionally categorized as CD8+ T cells in RCC, whereas DPT in other tumor types are mixture of CD4+ or CD8+ T cell subsets. We also found overlap of TCR profiles between DPT and CD8+ T cell subsets (Tex, ZNF683-CD8, and GZMK-CD8) in RCC. Together, multimodal single-cell analysis of TILs highlighted heterogeneity among tumor types that may provide insight into novel strategies to treat cancer.

Supported by a research grant from GlaxoSmithKline and the Providence Portland Medical Foundation.

Supported by a research grant from GlaxoSmithKline and the Providence Portland Medical Foundation.
Precise spatial multiplexing of immune cell diversity in clinical FFPE tumor samples with ChipCytometry™
Authors: Schwarz Spencer , Christians Arne , Brooks Jennifer , Northcutt Adam , Kwarta Karen , Winkeler Crystal
Published: January 2023

Highly multiplexed spatial biomarker analysis has demonstrated the potential to advance our current understanding of the immune system and its role in cancer – from tumor initiation to metastatic progression. Previously, a trade-off between plex and spatial context meant that our understanding of immune cell involvement in cancer was limited by either single-plex technologies with spatial context (i.e., immunohistochemistry) or highly multiplex technologies without spatial context (i.e., single-cell RNA sequencing). ChipCytometryTM is a novel, highly multiplexed technology that preserves both plex and spatial context to deeply profile immune cell diversity at single-cell resolution. ChipCytometry uses commercially available antibodies and combines iterative immuno-fluorescent staining with high-dynamic range imaging to profile dozens of protein biomarkers in a single tissue specimen. Cellular phenotypes are identified with via flow cytometry-like hierarchical gating from standard multichannel OME-TIFF images, compatible with a variety of computational tools being developed for multiplexed analysis and visualization. Here, we use ChipCytometry to identify and quantify key immune cell subtypes in both normal (i.e., appendix) and tumor tissues (i.e., melanoma and non-small cell lung cancer) FFPE samples. The results show precise expression levels for each biomarker in the assay in each individual cell in the sample, while maintaining spatial positioning of each cell. Spatial analysis reveals quantifiable heterogeneity of immune cell infiltration within the tumor samples, demonstrating the utility of the ChipCytometry platform for the in-depth immune profiling in clinical FFPE samples.
Defining the cooperation between T cell responses to MHC-I and MHC-II melanoma neoantigens towards developing effective personalized cancer immunotherapies.
Authors: Williams Charmelle , Shavkunov Alexander , Keshari Sunita , Salmon Avery , Highsmith Anna , Pineda Josue , Gubin Matthew
Published: January 2023

Immune checkpoint therapy (ICT) (e.g. anti-CTLA-4, anti-PD-1) enables durable T-cell dependent anti-tumor immunity in patients with solid tumors. Since not all patients respond to ICT, this work aims at developing a more in-depth understanding of T-cell responses to MHC class I (MHC-I) and MHC class II (MHC-II) tumor antigens that occur as a consequence of aberrant expression of non-mutant antigens or driver and passenger mutations that form tumor neoantigens. We used a poorly immunogenic Brafv600e Pten−/− Cdkn2a−/− YUMM1.7 (Y1.7) murine melanoma line that with a paucity of endogenous neoantigens that is unresponsive to ICT, and introduced model neoantigens previously identified in a murine sarcoma tumor line expressing the same MHC haplotype as the Y1.7 line. The MHC-I neoantigen used was formed by a G1254V point mutation in the Laminin alpha subunit-4 (Lama4) and the MHC-II neoantigen was formed by an A710T point mutation in Integrin beta-1 (Itgb1). Anti-CTLA-4-treated mice bearing the parental Y1.7 melanoma displayed progressive tumor growth, whereas the modified Y1.7 line expressing mLama4 and mItgb1 (Y1.7.mLama4.mItgb1) was rendered sensitive to ICT. Preliminary data showed that anti-CTLA-4 treated Y1.7.mLama4.mItgb1 tumors reject in mice in a CD4+ T cell and CD8+ T cell-dependent manner. Additionally, intratumoral mLama4-specific CD8+ T cells were detected in anti-CTLA-4-treated Y1.7.mLama4.mItgb1 melanoma bearing mice. Overall this model will allow us to better understand T cell mediated anti-tumor responses to both MHC-I and MHC-II neoantigens, as well as shared, non-mutant melanoma antigens (e.g. Pmel/GP100 and Trp2), which may facilitate development of improved neoantigen-based personalized immunotherapies.
Differential Immune Response to Klebsiella pneumoniae O-antigen subtypes O2v1 and O2v2
Authors: Wantuch Paeton L , Harding Christian M , Rosen David A
Published: January 2023

Klebsiella pneumoniae (Kp) infections are sharply on the rise among at-risk populations. We have previously shown that use of bioconjugate vaccines targeting Kp are beneficial in preventing disease and seek to increase our vaccine breadth of coverage. Kp has nine serogroups of O-antigens. Recently, additional O-antigen subtypes within these serogroups have been identified; yet, the pathogenic fitness, immunogenicity, functional antibody response, and cross-protection between subtypes is unknown. We investigated how the addition of the single branched galactose in O-antigen subtype O2v2 compared to O2v1 changes its bacterial fitness and host immune susceptibility. We knocked out the gmlABC region of an O2v2 strain of Kp, converting it to O2v1. Complementation of this mutant allowed us to identify the specific gene responsible for the addition of the single branched galactose of O2v2. Further, experiments using the O2v1 mutant and its parent O2v2 strain confirmed similar phenotypic expression of virulence factors beyond the O-antigen. Well-established murine models of pneumonia were used to determine the pulmonary fitness of the pathogens and assess the host innate immune responses. Neutrophil and complement-mediated killing assays suggested differences in susceptibility to innate immune defenses. Further, differences in IgG subclass were observed 4 weeks post respiratory infection with the O2v2 parent and O2v1 mutant strains. Lastly, using novel bioconjugate vaccines against these specific O-antigen subtypes, we determined only partial cross-protection is elicited. These studies advance our understanding of the immune response to Kp O-antigens and aid in vaccine design to combat this drug-resistant pathogen.

Supported by the W.M. Keck Postdoctoral Fellowship in Molecular Medicine Dr. Wantuch is a W.M. Keck Fellow
Effect of E-cigarette use on lung immunity to influenza infection
Authors: Cannon Judy L , Sciesczka David , Mrass Paulus , Garland Devon , Major Katharine , Hunter Russell , Begay Jessica , Lucas Selita , Campen Matthew J
Published: January 2023

Use of E-cigarettes, also called vaping, has been significantly increasing, with over 10 million adults and 3 million adolescents reported to actively vape. Of particular concern recently is the association of vaping with increased risk of respiratory infection, including influenza and COVID-19. Despite the claims of safety, lung immunological development appears to be impacted by vaping, with associated pulmonary toxicity and potential to impair immunity against respiratory infections.

Immunity against respiratory pathogens, particularly viral pathogens is dependent on a robust CD4 and CD8 T cell responses responsible for viral clearance. CD4 and CD8 T cells then develop memory responses to protect against reinfection. T cell memory also provides protection against infection after vaccination. We asked whether vaping exposure affects T cell responses to influenza infection. We exposed animals for 8 weeks to base components of vape devices, including propylene glycol (PG), vegetable glycerin (VG), and a novel cutting agent, phytol, then assessed immune responses to influenza. We find that PGVG and phytol exposure alters pulmonary function, promotes proteomic changes to the lung, and modify immune cell subsets in the lung. We also find that PGVG and phytol exposure alters T cell responses to influenza infection, particularly T cell memory protection against heterologous infection. These results suggest that vaping affects immune responses to respiratory influenza infection, including T cell memory responses. Effects of vaping on T cell memory may have wide ranging consequences for T cell mediated protection against other respiratory disease including SARS-CoV-2 and protection after vaccination.

Supported by NIH (P20 GM121176; P20GM130422)
Human lung tissue-resident memory CD8+ T cells are transcriptionally, epigenetically, and phenotypically diverse
Authors: Elliott Jennifer L , Kost Kirsten N , Lacetti Kristiana H , Thomas Jeronay K , Williams M Elliott , Mattingly Cameron L , Lobby Jenna L , Lawrence Laurel A , Kohlmeier Jacob E
Published: January 2023

Tissue-resident memory CD8+ T cells (TRM) are strategically located in peripheral tissues, especially at mucosal surfaces, where they can provide protection against invading pathogenic microbes. In the lungs, TRM play a critical role in limiting disease and transmission of respiratory viruses; however, the heterogeneity present in TRM populations in the human lung remains largely unexplored. Here we show that human lungs harbor transcriptionally, epigenetically, and phenotypically distinct populations of memory CD8+ T cells expressing the tissue residency-associated markers CD69 and CD103. High-dimensional flow cytometry and single-cell RNA sequencing of memory CD8+ T cells isolated from human lungs shows that heterogeneity exists both between and within CD69+ CD103− and CD69+ CD103+ subsets, with transcriptional diversity among the CD69+ CD103− subset being most prominent. Single-cell ATACseq demonstrates that similar levels of epigenetic diversity exist between and within these different TRM subsets. However, flow cytometry and single-cell RNAseq of influenza- and SARS-CoV-2-specific lung TRM demonstrates that heterogeneity between CD69+ CD103− and CD69+ CD103+ subsets is largely eliminated when the cells are specific for a common antigen. Together, these data illuminate underappreciated and unexplored aspects of heterogeneity in TRM populations in humans.

Supported by grants from NIH/NIAID (75N93019R00028) and NIH/NHLBI (R35 HL150803)
CD4 T cell deficiency results in impaired neutralizing IgG against polyomavirus carrying a frequent PML-associated capsid mutation
Authors: Ayers Katelyn Nicole , Lauver Matthew D , Lukacher Aron E
Published: January 2023

Deficiencies in the CD4 T cell compartment, such as HIV/AIDS, idiopathic CD4 lymphopenia, predispose patients to progressive multifocal leukoencephalopathy (PML), an oft-fatal demyelinating brain disease caused by the JC polyomavirus (JCPyV). PML is associated with immunomodulatory therapies that preferentially diminish CD4 T cells in the CSF (natalizumab) or B cells (rituximab). The connection between CD4 T cells and JCPyV control is unclear. Work by others show that PML patient sera have “blind spots” in neutralizing JCPyV variants with mutations in the VP1 capsid protein. We hypothesized that defects in the humoral response after CD4 T cell depletion led to the emergence of antibody (Ab)-escape PyV variants. Using mouse PyV (MuPyV) to model JCPyV CNS disease, we found immune cell aggregates consisting of B cells, CD4 T cells, and CD8 T cells along the ventricles of PyV-infected WT mice but only a few infected cells. In contrast, CD4 T cell-depleted mice had more infected ependyma but lacked lymphocytic aggregates. There were fewer anti-PyV Ab-secreting B cells in the CNS despite similar B cell numbers in MuPyV-infected WT and CD4 T cell deficient mice brains. CD4 T cells prevent emergence of VP1 mutant PyVs that escape neutralizing Abs. MuPyV infection elicits a CD4 T cell-independent anti-VP1 IgG response that neutralizes WT virus equivalently to IgG from CD4 T cell-sufficient mice. Serum from MHC-II KO and CD4 mAb depleted mice had significantly lower neutralization activity toward a MuPyV with a frequent PML-equivalent VP1 mutation than to WT MuPyV. This data demonstrate that a narrowed antiviral IgG response caused by CD4 T cell deficiency is a critical antecedent for outgrowth of Ab-escape PyV VP1 mutant viruses.

Supported by 1. Finkelstein Memorial Student Research Award, Pennsylvania State University College of Medicine, Hershey, PA 2. T32 CA060395 Training Grant (Meyers), NIH/NCI 3. R01 NS088367, NIH/NCI
IFN-γ promotes TH1 at the expense of TFH differentiation during viral infections
Authors: Sala Eleonora , Mangione Marta , Laura Chiara , Consolo Eleonora , Beccaria Cristian Gabriel , Iannacone Matteo , Kuka Mirela
Published: January 2023

Although humoral and cellular immunity upon viral infections usually co-exist, sometimes one of the two responses emerges and is responsible for most of the antiviral activity. For example, vescicular stomatitis virus (VSV) infection induces early and potent neutralizing antibody (nAb) responses, whereas lymphocytic choriomeningitis virus (LCMV) infection induces strong cellular responses, but weak nAb responses. Recent work from our laboratory has shown that unbalance is observed also at the level of CD4 T cells responses, with VSV inducing strong TFH polarization that support nAb responses, and LCMV in contrast promoting TH1 differentiation. Analysis of the VSV and LCMV priming niches led to identification of the spatiotemporal regulation of type I IFN expression as a critical regulator of antiviral CD4+ T cell polarization, with early type I IFN sensing leading to TFH polarization. Notably, this mechanism did not explain the strong TH1 differentiation observed during LCMV infection. In this study we aimed to elucidate the role of known TH1-polarizing cytokines in CD4+ T cell differentiation upon LCMV infection. We found that IFN-γ, but not IL-12, plays a key role in early CD4+ T cell differentiation upon LCMV infection, inducing TH1 cell polarization and suppressing TFH cell development, thus resulting in a shift in the equilibrium towards TFH and humoral responses. Future studies will determine the cellular source for this TH1-polarizing cytokine and the mechanism by which IFN-γ exerts its function, possibly unveiling the mechanisms underlying the reduced humoral response in the context of viral infections like LCMV.
Immunogenetic determinants of HSV-2 infection and disease
Authors: Graham Jessica B , Swarts Jessica , Mooney Michael , Lund Jennifer M
Published: January 2023

Herpes simplex virus-2 (HSV-2) is one of the most prevalent sexually transmitted infections, and can result in life-long, chronic disease. Disease severity, frequency of reactivation, and shedding rates vary between individuals, though little is known about how host genes regulate tissue-specific immune responses. We have previously used the Collaborative Cross (CC) mouse model system, which incorporates the extent of genetic variation found in the human genome, to better model the diversity of outcomes found in human viral infections, so we next probed the CC to identify host genetic regions that regulate viral shedding and disease following HSV-2 infection, as well as tissue-specific immune responses. We performed a screen of mice from different CC strains to assess viral titers and disease following vaginal HSV-2 infection, and then used this data to perform quantitative trait loci (QTL) mapping to identify chromosomal regions linked to vaginal viral shedding rates and levels, as well as virus-associated clinical disease. In parallel experiments, we assessed lymphoid, nervous system, and mucosal immune cell frequencies at innate, adaptive, and memory response timepoints. We observed a distinctive suppressive signature on vaginal Tregs compared to lymph node Tregs, at various times following HSV-2 infection. Additionally, these suppressive responses varied in mice with either higher viral titers, or more tissue inflammation, highlighting the interplay between host immune response and viral infection kinetics. Understanding host factors that contribute to HSV shedding, clinical disease, and immune responses may provide critical insights for developing new preventive strategies or interventions to HSV-2 infection.

Supported by grant R21 AI152559-01 from the NIH
EVALUATION OF Murine norovirus (MNV)-1 INFECTION IN PRIMARY MOUSE B CELLS.
Authors: Rodriguez Carlos Emilio Miguel , Lonches Clotilde Cancio , Escolano Ana Lorena Gutierrez , Argumedo Leopoldo Santos
Published: January 2023

Methods

Primary mouse spleen B cells were stimulated or not with LPS (10ug/ml) + IL-4 (10IU/ml) for 48 hours. Two million cells were infected 72 h with MNV-1 at a MOI of 5. The non-structural (NS7) viral protein expression was analyzed in cell lysates by Western blot with a specific anti-NS7 antibody. The supernatant-associated virus in Raw 264.7 cells was determined by paque assay.

Results

B cells can be infected by MNV-1, even though they do not express the functional receptor CD300LD and CD300LF present in macrophages and Tuft cells. Since MNV-1 can uses CD44 as a receptor in BMDCs, we wanted to investigate if this molecule has a role in MNV-1 entry into B cells. First, we corroborated MNV-1 infection of B cells by evaluating the presence of NS7 by western blot and viral particles production in the supernatant at 72 hpi. To evaluate the role of CD44 in MNV-1 entry into B cells, we blocked the CD44 receptor using two different anti-CD44 monoclonal antibodies, and we found a significant decrease in both the amount of NS7 protein and virus yield, indicating that CD44 plays a role during virus binding/entry into B cells. Since CD44 expression is increased in LPS + IL-4 activated B cells, we wanted to determine if MNV-1 infection could be favored in these conditions. A correlation between the activation of B cells for 48 h and a significant increase of twice of MNV-1 NS7 protein and virus yield production were detected after infection.

Conclusion

Blocking CD44 correlates with a reduction of MNV-1 protein synthesis and virus yield production, indicating a role of CD44 in MNV-1 binding/entry. The increase of MNV NS7 protein and virus yield production in C57BL/6J spleen B cells activated with LPS + IL-4 that overexpresses CD44 on its surface, also supports this conclusion.

CEMR is supported by a Conacyt fellowship, CVU fellowship number: 780860
The role of tumor necrosis factor receptor 2 in experimental autoimmune encephalomyelitis
Authors: Bartsch Brenden L , Negron Austin Joseph , Forsthuber Thomas
Published: January 2023

Multiple sclerosis (MS) is an autoimmune neuroinflammatory disease that affects over 2 million people globally. HLA-DR2b is a major histocompatibility complex (MHC) locus in humans responsible for antigen-specific immune responses. HLA-DR2b (DRB1*15:01) is a major genetic risk factor for MS. Experimental autoimmune encephalomyelitis (EAE) is the animal model for MS and can provide insights into the mechanisms behind MS pathogenicity. Tumor necrosis factor receptor 2 (TNFR2) was suspected to control EAE severity by altering regulatory T cell (Treg) function and impairing remyelination. In the absence of TNFR2, mice expressing the human DR2b allele and lacking mouse MHC II develop progressive EAE upon adoptive transfer of myelin oligodendrocyte glycoprotein (MOG)-reactive CD4 T cells. These mice show altered Treg phenotype, including decreased expression of Helios, a marker of thymic Tregs, by splenic Tregs from DR2bΔR2 mice compared with DR2b mice. However, it remains unclear how the presence or absence of TNFR2 alters the expression of specific protective genes in both astrocytes and Tregs in the context of EAE. Using the DR2bΔR2 and DR2b mouse models, we investigated the impact of TNFR2 on protective astrocyte subsets and regulatory T cells by single-cell RNA sequencing (scRNA-seq) in the context of both active and adoptive transfer EAE. Our results elucidate mechanisms underlying the protective function TNFR2 via modulating astrocyte and regulatory T cell function in EAE. Our results may aid in the development of new treatments for MS.

Supported by grant 1RO1NS117742 from the NIH/NINDS (T.G.F.) and by NIH/NIGMS RISE grant GM60655 (BB).
IL7Rα and TSLPR expression on Langerhans cells in response to inflammatory stimulation
Authors: Gonzalez-Rodriguez Martin Ignacio , Salomaa Tanja , Kummola Laura , Hiihtola Lotta , Junttila Ilkka
Published: January 2023

A key component of immune barriers are innate immune cells localized in the epithelium responsible for sensing local signals such as foreign antigens and cytokines. In the skin, specialized antigen presenting cells (APC) known as Langerhans cells (LC) can integrate these signals influencing downstream T cell activation and polarization. Thymic stromal lymphopoietin (TSLP) is a proinflammatory mediator released by keratinocytes that promotes LC to enhance type 2 immune response (Th2). When Th2 response becomes unbalanced, it induces type 2 inflammation as seen in atopic dermatitis. TSLP signaling is regulated by two cell surface receptor chains: TSLP receptor (TSLPR) and IL7Rα. Interestingly, IL7Rα is not commonly expressed in murine conventional APC such as dendritic cells (DC). Our previous findings have shown that IL7Rα expression regulates TSLP sensitivity of splenic DCs (sDC) ex vivo. However, the receptor expression dynamic in tissue resident LC and its biological implications has not yet been elucidated. In this study we enriched LC from healthy mice B6 mice using MACS magnetic beads. Cytokine surface receptors and pospho-STAT5 activation were assessed using flow cytometry. Moreover, topic administration of vitamin analog MC903 will be used to evaluate TSLPR and IL7Rα dynamic. Our current results indicate that IL7Rα is expressed on unstimulated LC contrary to sDC. Furthermore, TSLPR was not differently expressed between LC and sDC. Functional assessment of p-STAT5 ex vivo and the dynamic of these receptors in the context of MC903 treatment in vivo is yet to be measured. Our results suggest that tissue resident APC regulate differently cytokines receptors enabling spatial-temporal regulation of proinflammatory signals.

Academy of Finland (IJ: grants: 25013080481 and 25013142041), The Competitive State Research Financing of the Expert Responsibility Area of Fimlab Laboratories (grant X51409 to IJ), Tampere University Hospital Support Foundation (IJ) and Tampere Tuberculosis Foundation (IJ).
Regulation of IL-2- and IL-6-dependent signaling in human Tregs by CISH and SOCS3
Authors: Vujanac Milos , Ding Ying , Malek Thomas R
Published: January 2023

SOCS proteins function as negative regulators of cytokine signaling. However, the role in of these proteins in human Tregs remains poorly understood. To address this issue, CRISPR-Cas9 genome editing was used to probe the role of SOCS3 and CISH in ex vivo expanded human Tregs. High efficiency editing of SOCS3 and CISH was confirmed at the DNA and RNA levels and the protein level for CISH. Deletion of SOCS3 resulted in sustained activation of STAT3 upon treatment with IL-6 whereas IL-2-induced STAT5 activation and IL-10- induced STAT3 activation were not affected. These results suggest that in Tregs SOCS3 specifically targets signaling initiated through IL-6R. Proliferative capacity and expression of Treg-specific phenotypic markers were not affected by SOCS3 ablation. RNA-seq revealed a common transcriptional signature between IL-6-treated wild-type and SOCS3-edited cells, showing upregulation of pro- and anti-inflammatory response genes and other genes that may affect Treg stability. Other differentially expressed genes in SOCS3-edited cells belong to pathways that may play a role in how Tregs integrate cytokine signals. In contrast, CISH editing resulted in increased IL-2-dependent activation of STAT5 and STAT3 and led to increased proliferation of Tregs. These effects were accompanied by only minor change in the expression of Treg-specific phenotypic markers. Our data indicate a restricted role of SOCS3 and CISH in regulating the responses of Tregs to IL-6 and IL-2, respectively. These activities likely contribute to maintaining Treg homeostasis under normal and inflammatory conditions.

Supported by: NIH R01AI131648
Characterization of anti-porcine CXCL10 monoclonal antibodies
Authors: Lunney Joan K , Hailstock Taylor , Dai Chaohui , Aquino Jovan , Chick Shannon , Walker Kristen , Manirarora Jean N , Gourapura Renukaradhya J , Sullivan Yvonne B , LaBresh Joanna
Published: January 2023

C-X-C motif chemokine ligand 10 (CXCL10) facilitates chemoattraction of important immune cells to tissues. As part of the USDA-NIFA Swine Immune Toolkit Initiative, our goal is to provide the veterinary community with new commercial immune reagents and standardized assays for future research efforts. For the chemokine target CXCL10, we used yeast expressed, recombinant porcine CXCL10 (rPoCXCL10) to generate a panel of α-CXCL10 monoclonal antibodies (mAbs). Each of the α-PoCXCL10 mAbs was assessed by ELISA using cross-inhibition analyses of biotinylated mAbs, and direct binding to orthologous yeast expressed CXCL10 proteins. As a result, we assigned six distinct epitope groups for the nine generated α-CXCL10 mAbs. Subsequently, we screened AF647-tagged α-CXCL10 mAbs for intracellular staining of pig immune cells using different stimulation conditions. Of the nine mAbs only two detected intracellular CXCL10 expression in PMA/ionomycin or rPoIFNγ-stimulated porcine cells. Further, cell characterization assays verified CXCL10+ cells as CD3-CD4-CD172+, with occasional CD4+ subsets. Overall, we have determined that α-CXCL10-1.4 mAb is the best mAb clone for intracellular staining and recommend it for use in analysis of intracellular signaling to detect interactions that regulate cell migration. A sandwich ELISA was also developed to quantitate CXCL10 protein expression; it verified reactivity with native porcine CXCL10. Overall, these tools will be useful for evaluating immunity against swine infectious diseases and host-responses to vaccines. New reagents identified by the Swine Immune Toolkit Initiative will undoubtedly advance future swine research efforts. Supported by USDA-NIFA AFRI grant # 2019-67015-29815.

Supported by USDA-ARS project # 8042?32000?102 and USDA-NIFA AFRI grant # 2019-67015-29815
Chemokines and anionic phospholipids: new binding partners for microbial killing and apoptotic cell clearance
Authors: Pontejo Sergio , Murphy Philip
Published: January 2023

Chemokines constitute a large family of small cytokines that regulate leukocyte migration in immunity and inflammation, some of which also have direct bacteria-killing properties. While the molecular mechanism by which chemokines kill bacteria remains poorly understood, chemokines mediate cell migration by interacting with cell surface glycosaminoglycans (GAGs) and G protein-coupled receptors (GPCRs). We have discovered that many chemokines interact with high affinity with anionic membrane phospholipids, including phosphatidylserine (PS) and cardiolipin (CL) which are selectively exposed on the membrane of apoptotic cells and apoptotic extracellular vesicles (ApoEV), and bacteria, respectively. This adds to GPCRs and GAGs a third class of cell surface binding site for chemokines and expands the scope of chemokine action to the biological niches occupied by PS and CL. For instance, we found that ApoEV induce phagocyte migration for apoptotic cell clearance by presenting PS-binding chemokines on the vesicle surface to GPCRs expressed by phagocytes. This is a novel PS-dependent mechanism for chemokine delivery by extracellular vesicles potentially relevant to other areas of immunology and indicates that chemokines may play a more prominent role in the removal of dying cells than previously reported. On the other hand, we discovered that only CL-binding chemokines are antimicrobial and that CL-deficient bacteria are resistant to chemokine treatment, indicating that chemokines kill bacteria by targeting CL on the bacterial membrane. In short, we have identified chemokines as the first family of soluble cytokines whose bioactivities can be regulated by direct interaction with membrane phospholipids.

Supported by NIAID/NIH intramural research program
IL-18R signaling impairs hematopoietic recovery after severe, shock-like infection
Authors: Howard Jennifer E , MacNamara Katherine C
Published: January 2023

Severe infection dramatically alters blood production, but the mechanisms impacting hematopoietic stem and progenitor cell (HSC/HSPC) recovery have not been carefully investigated. Using Ixodes ovatus Ehrlichia (IOE), a pathogen that causes severe shock-like illness and bone marrow (BM) aplasia, we explored the kinetics and mechanisms of hematopoietic recovery. Doxycycline treatment is necessary to prevent IOE-induced death and control bacterial burden in mice, but is not sufficient to prevent HSC/HSPC loss. During recovery from acute infection (12dpi) we saw increased cellularity of LT-HSCs and MPPs, accompanied by diminished function measured by decreased colony forming units (CFUs) in methocult assays. Reduced CFUs indicate a deficiency in BM myelopoietic function during recovery phase post- IOE. We previously showed that IL-18R-deficient mice exhibited improved CFUs at 12dpi, suggesting IL-18R signaling is detrimental to recovery from shock-like IOE. Utilizing a mixed BM chimeric mouse model (50:50 WT:IL-18R−/−), we observed that during recovery at 15dpi, IL-18R−/− cellularity predominates the LT-HSC and MPP2 populations, demonstrating an intrinsic role for IL-18R in limiting recovery of these populations. Additionally, BM chimeras at 15dpi showed increased levels of cell death (Annexin V+ and 7AAD+) in WT HSCs compared with IL-18R−/− HSCs. Transplantation of whole BM from mixed BM chimeric mice revealed a pronounced bias for IL-18R−/− cells, indicating that IL-18R−/− cells had a competitive advantage and improved function after IOE infection. Together, these data support the conclusion that IL-18R signaling impairs HSC function during recovery from severe, shock-like infection via decreased HSC viability.

This work was supported by National Institutes of Health funding from NIGMS (R35GM131842) to KCM.
IL-7 and IL-7R regulate fetal macrophage establishment via survival and proliferation
Authors: Leung Gabriel , Valencia Clint , Krum Brian , Beaudin Anna E
Published: January 2023

We have recently reported that IL7R signaling regulates fetal macrophage development, in addition to lymphocyte lineage commitment and development. Specifically, IL7R deletion and pharmacological blockade during a defined fetal developmental window impairs the establishment of fetal macrophage populations in the liver, lung, brain, and epidermis. We next hypothesized that IL-7 is also required for fetal macrophage establishment and that IL-7 signaling activates developmental programs that facilitate macrophage development via proliferation and survival. Immunofluorescent staining of E14.5 murine embryos revealed abundant co-localized IL7R and F4/80 expression in fetal liver, lung, brain, and skin. IL7R+ F4/80-expressing cells were also found in close proximity to IL-7+ cells, suggesting they participate in IL-7 signaling. Moreover, germline deletion of IL-7 also resulted in postnatal depletion of fetal-derived macrophage populations. Antibody blockade of IL-7R receptor during late gestation revealed tissue-specific deficits in survival and proliferation the day before birth. Specifically, liver and lung macrophages showed increased apoptosis, as determined by Annexin V staining, while brain and epidermal macrophages were less proliferative, as determined by Ki67 staining. Here we have shown that IL-7, in addition to IL7R, regulates establishment of tissue-resident macrophages and that fetal blockade of IL7R signaling regulates establishment by impairing survival and proliferation during late gestation. Ongoing work seeks to define IL7/IL7R signaling pathways in developing macrophages, as well as use genetic models to pinpoint the requirement for IL7R in fetal myeloid specification.

Supported by grants from NIH (R01 10057561).
Age Associated B Cells positively associate with atherosclerosis in mice and Humans
Authors: Srikakulapu Prasad , Pattarabanjird Tanyaporn , Bontha Sai Vineela , Upadhye Aditi , Drago Fabrizio , Marshall Melissa , McNamara Coleen
Published: January 2023

Age-Associated B cells (ABCs: CD19+ CD11c+) have recently been reported as a new subset of B cells with unique cell surface and transcriptional signatures. ABCs are mostly comprised of antigen experienced memory B cells that arise in response to microbial infections. ABCs have been reported to be elevated in autoimmune diseases, where in, these cells have characteristics of auto-antibody producing memory B cells. However, the role of ABCs during atherosclerosis has not be studied. Atherosclerosis is a chronic inflammatory disease and the major underlying cause for cardiovascular diseases (CVD). We for the first time report the role of ABCs in both atherosclerotic mice and in human CVD. Chow fed 50- and 100- week old, male, ApoEKO mice were used to study atherosclerosis. Lesion area was measured following Sudan-IV enface staining of aorta. 100-week-old mice developed significantly more disease than the 50-week-old mice. Further, through flow cytometric analysis ABCs were detected in spleen, bone marrow (BM) and blood of these mice. The frequency of ABCs from total B cells was significantly higher in all these tissue compartments in 100-week-old mice compared to 50-week-old mice. Additionally, most ABCs (>80 %) were actively proliferating cells (Ki67+), as compared to normal CD11c− B cells (~35 %). In mice, we observed a significant correlation of atherosclerosis disease levels with the frequency of ABCs in spleen (p=0.004, R2=0.61), BM (p=0.01, R2=0.53), and blood (p=0.0005, R2=0.75). In humans as well, the frequency of circulating ABCs was significantly higher in CVD subjects compared to non-CVD subjects. Results from this study suggest a strong association of ABCs with aggravated atherosclerosis in both murine models and humans with CVD.
Arterial antibodies promote atherosclerotic plaque development in mice
Authors: Taylor Joshua A , Hutchinson Mark A , Gearhart Patricia J , Maul Robert W
Published: January 2023

Atherosclerosis is characterized by the progressive buildup of cholesterol-rich plaques within the arterial wall. Plaques activate the immune system, of which B cells play a major role. Antibodies bind autoantigens released from necrotic cells and form immune complexes to increase inflammation. We isolated aorta-associated B cells from atherosclerotic ApoE-deficient mice on a high-fat diet and performed single cell sequencing of the B cell receptors to obtain antibody pairs. 32 over-represented antibodies were identified and then expressed in Expi293F cells. Epitope determination of these antibodies was performed against a human proteome array, and 15 potential autoantigens bound the monoclonal antibodies with significant affinity and specificity. The 12 most-promising antigens were scrutinized for their location in plaque tissue by immunohistochemistry using the cognate antibodies. Nine of these relevant antigens were then successfully synthesized using the pET bacterial expression system, and purified proteins were used to interrogate antibodies in human and mouse sera by ELISA. Mice were then immunized with the 5 best antigens to confirm their immunogenicity. The relevant antigens and antibodies are now being tested to see if they will protect or promote atherosclerosis development, and if they can be used as biomarkers in human disease.

This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging.