bluuebunny/crossref_metadata_2025 huggingface dataset:
Combining Immunotherapies with Conventional Cancer Therapies in a Preclinical Model of Treatment-Resistant, High-Risk Neuroblastoma
Authors: Zebertavage Lauren K , Aiken Taylor , Erbe-Gurel Amy , Schopf Allison , Nielsen Megan , Katz Sydney , Rakhmilevich Alexander , Sondel Paul
Published: January 2023

Patients diagnosed with neuroblastoma (NBL) are segmented into categories, with ~50% considered high-risk and ~40% of those patients eventually succumbing to disease. Our group published a regimen (“CAIR”) combining adaptive and innate immunotherapies with radiotherapy (RT) to treat an immunologically cold, treatment-resistant model of NBL (9464D-GD2). Despite CAIR being our most effective treatment, it only cures ~40% of tumors and lacks clinical relevance due in part to toxicities. Here we aimed to test if the clinically-approved salvage therapy of temozolomide and irinotecan (T/I) and αGD2-based therapy replicated human trials and to test if CAIR can be reduced to improve relevance.

For chemo studies, mice were treated T/I and αGD2 immunocytokine (Hu14.18-IL2). T/I or Hu14.18-IL2 alone did not extend survival of 9464D-GD2-bearing mice (p=0.596 and p>0.999). T/I and Hu14.18-IL2 together extended survival (p=0.036 and p=0.004) but none of these treatments resulted in cures. To test CAIR reductions, mice were treated with RT, αCD40, αCTLA4, CpG, and Hu14.18-IL2. The full regimen cured 36% of mice (8/22), CAIR minus αCTLA cured 43% (9/21), CAIR minus αCD40 cured 50% (8/16), and CAIR minus CpG cured 44% (7/16). CAIR minus RT or Hu14.18-IL2 cured no mice (0/8 each).

Demonstrating similarity to human NBLs that do not respond to salvage treatment of T/I and αGD2 therapy, 9464D-GD2 tumors are not cured by T/I and αGD2 immunocytokine (Hu14.18-IL2). In contrast, CAIR can cure 9464D-GD2 bearing mice, and our data indicate that this regimen can be further reduced without decreasing efficacy. A reduced regimen enhances both clinical relevance and our ability to add additional agents to further improve survival.

Fellowship support provided by the Hartwell Foundation.
Hallmark features of T cell dysfunction are established within hours after tumor antigen encounter
Authors: Rudloff Michael William , Favret Natalie , Zumbo Paul , Dündar Friederike , Betel Doron , Philip Mary
Published: January 2023

Tumor-specific CD8 T cells (TST) found in patients with cancer are dysfunctional and unable to halt cancer progression. TST dysfunction, also known as exhaustion, is encoded by specific epigenetic and transcriptional programs and thought to be induced by chronic antigen stimulation. Using a genetic liver cancer mouse model, we followed the fate of naïve TST in tumor-bearing hosts. Strikingly, we found that within hours after encountering tumor antigen, TST lost effector function, expressed high levels of inhibitory receptors, and displayed chromatin peak accessibility changes previously characterized in late-stage dysfunction. This rapid onset challenges the paradigm that chronic antigen/T cell receptor stimulation is required to induce dysfunction/exhaustion. Moreover, dysfunction-associated epigenetic features were established even prior to cell division, opposing the prevailing notion that cell divisions are required for epigenetic remodeling and T cell differentiation. To determine whether these early dysfunction hallmarks were “imprinted”, we reisolated TST from malignant lesions and transferred them into antigen-free hosts. While loss of effector function was imprinted within hours of tumor exposure, other dysfunctional hallmarks, including PD1 expression, required several days of tumor exposure and additional chromatin remodeling to become stably encoded. Our study reveals the rapid induction and epigenetic remodeling which underlies TST commitment to the dysfunctional fate, highlighting the need to target early activation-induced pathways to improve anti-cancer T cell immunity.

Supported by T32GM007347-43
Directing T cell alloreactivity against solid tumors through tumor antigen dependent TCR expression
Authors: Christie Karen Michelle , Zhang Shenyu , Schwarz Thomas A , Ma Zhengyu
Published: January 2023

A major challenge in T cell therapy against solid tumors is the heterogeneity of tumor antigen (TA) expression. Elimination of TA+ tumor cells often fails to suppress tumor growth due to the expansion of TA− tumor cells. To address this, we developed tumor-activated alloreactive T cells (TAATs) that display alloreactivities only in tumors. TAATs lack endogenous CD3ɛ expression and contain a tumor sensing mechanism that drives the expression of an exogenous copy of CD3ɛ in the presence of TA+ tumor cells. TAATs are therefore inactive in normal tissues due to retention of TCR/CD3 in the ER but become alloreactive in tumors. TAATs attack all HLA+ cells in tumors including tumor cells, stromal cells and cells that suppress immune functions. To generate TAATs, HLA-mismatched allogeneic T cells were stimulated with HLA class I+ breast cancer cells to expand alloreactive T cells. Endogenous CD3ɛ expression was then knocked out using CRISPR/Cas9. The T cells were also modified to express a Her2-specific synthetic Notch receptor that releases an intracellular domain containing a Gal4-VP64 transcription activator upon receptor engagement. In addition, an expression cassette with a CD3ɛ coding sequence driven by a promoter containing Gal4 binding sites was introduced to the T cells. When co-cultured with Her2+ cancer cells, TAATs restored TCR/CD3 expression and were able to degranulate and produce IFNγ in response to anti-CD3 stimulations. TAATs were also able to kill Her2+ cancer cells in co-cultures and kill Her2− cancer cells in the presence of Her2+ cancer cells. Compared with CAR T cells, TAATs may achieve superior tumor suppression through attacking a wider range of cells in tumors and by overcoming the immunosuppressive tumor microenvironment.
Identifying Key Differences Between Responders vs. Non-Responders to Immune Checkpoint Blockade for Metastatic Osteosarcoma (mOS)
Authors: Di Palma Michelle Pina , Appel Nicole , Lussier Danielle , Blattman Joseph N
Published: January 2023

Osteosarcoma is the most common childhood bone malignancy, with a sharp decline in survival rates upon metastasis. We have previously shown immune checkpoint blockade (ICB) of anti-CTLA-4/anti-PD-L1 for mice inoculated with a K7M2 metastatic osteosarcoma (mOS) cell line resulted in ~50% survival with complete tumor clearance, including recent analogous results upon replication of this treatment regimen. Differences in response rates to ICB are common among patients with the same malignancy across various cancers. Unlike inbred lab mice, human patients have diversity in genetic makeup and other factors, causing the discrepancy in ICB response to remain poorly understood. A culprit for these discrepancies is variance in circulating antibodies (Abs), which can indicate differences in disease presence, microbiome composition, or immune-modulating factors. Recent studies have characterized Ab patterns in sera by using microarrays with thousands of randomly sequenced peptides and incubating on them diluted sera. The Ab binding pattern on the microarray is termed an “immunosignature.” We have found that before inoculation with mOS or ICB, blood from mice displays distinct differences in immunosignatures between responders and non-responders, suggesting that the presence or absence of particular Abs may influence the outcome of ICB for mOS. Additionally, recent studies have discovered that ICB’s effectiveness can be associated with or even reliant on ICB recipients’ microbiome composition and that changes in microbiome composition can result in subsequent changes to circulating Abs. Here we analyze both differences in Ab composition and microbiome composition, highlighting their impact on ICB efficacy for mOS.

Funding support for this work has been provided by a grant through the Sarcoma Foundation of America (SFA). Additional funding support for this work has been provided by a grant through the Graduate and Professional Student Association (GPSA) at Arizona State University.
Improved anti-tumor T cell generation using rapidly differentiated dendritic cells
Authors: Minguy Annabelle , Trottier Jessica , Sanchez-Dardon Jaime , Bastien Jean-Philippe , Dave Vibhuti , Roy Denis Claude
Published: January 2023

After allogeneic stem cell transplantation, minor histocompatibility antigens (MiHAs), Wilms-Tumor 1 (WT1) antigen, and other peptides presented by host hematologic cancer (HC) cells can lead to the in vivo expansion of donor T cells crucial for the graft-versus-leukemia effect. While the specific ex vivo production of such leukemia-specific T cells represents an interesting treatment approach, the generation of these antigen-specific donor T cells represents a major challenge. We have developed a protocol enabling the infusion of monocyte derived dendritic cell (DC)-induced ex vivo expanded T cells specific for MiHAs, WT1 and other peptides that are preferentially expressed on hematologic cells for the treatment of relapsed HC patients. While it provides interesting results, our 42-day expansion protocol is costly and requires prolonged culture periods during which patient with HC could deteriorate rapidly. To accelerate T cell production, we compared DCs generated in 3 days (DC3) vs 9 days (DC9) using standard maturation cocktail and toll-like receptor agonist. DC3 showed small differences in expression of maturation/activation markers and a unique gene signature vs DC9. Importantly, the frequency of WT1-specific CD8 T cells, primed by DC3, was slightly higher than that in DC9 primed cultures. Upon restimulation, these DC3-primed-WT1-specific T cells expressed higher amounts of IFNγ, TNFα, and CD107a degranulation marker, and were cytotoxic against WT1-expressing autologous blast cells. Together, these data show that matured DC3 have a strong capacity to prime and expand functional anti-tumor antigen-specific CD8 T cells and thus offer most interesting features for ex vivo expansion of T cells for cancer immunotherapy.
IL-9/STAT3/fatty acid oxidation mediated lipid peroxidation contributes to Tc9 cell longevity and enhanced antitumor activity
Authors: Xiao Liuling , Yi Qing
Published: January 2023

CD8+ T cell longevity regulated by metabolic activity plays important roles in cancer immunotherapy. In vitro polarized IL-9-secreting CD8+ Tc9 cells exert greater persistence and antitumor efficacy than Tc1/CTL cells after adoptive transfer, but the underlying mechanism remains unclear. Here, we show that tumor-infiltrating Tc9 cells display significantly lower lipid peroxidation than Tc1 cells in several mouse models, which is strongly correlated with their persistence. Using RNA-sequence and functional validation, we found that Tc9 cells exhibited unique lipid metabolic programs. Tc9 cell-derived IL-9 activated STAT3, upregulated fatty acid oxidation and mitochondrial activity, and rendered Tc9 cells with reduced lipid peroxidation and resistant to tumor or reactive oxygen species (ROS) induced ferroptosis in TME. IL-9 signal deficiency, inhibiting STAT3 or fatty acid oxidation increased lipid peroxidation and ferroptosis of Tc9 cells, resulting in impaired longevity and antitumor ability. Similarly, human Tc9 cells also possessed lower lipid peroxidation than Tc1 cells and tumor-infiltrating CD8+ T cells expressed lower IL-9 and higher lipid peroxidation- and ferroptosis-related genes than circulating CD8+ T cells in melanoma patients. This study indicates that lipid peroxidation regulates Tc9 cell longevity and antitumor effects via IL-9-STAT3-fatty acid oxidation pathway and regulating T cell lipid peroxidation can be used to enhance T cell-based immunotherapy in human cancer.

This work was supported by NCI R01s CA200539 and CA239255, and Cancer Prevention & Research Institute of Texas Recruitment of Established Investigator Award (RR180044).
Optimization of expansion techniques for adoptive NK cell transfer in dogs with cancer
Authors: Razmara Aryana , Judge Sean J , Dunai Cordelia , Murphy William J , Rebhun Robert B , Kent Michael S , Canter Robert J
Published: January 2023

Natural killer (NK) cells can recognize heterogeneous cancer cell targets without prior sensitization, making them promising prospects for use in immunotherapy. We have completed first-in-dog feasibility clinical trials in dogs with cancer using both autologous and allogeneic NK cells expanded from peripheral blood mononuclear cells (PBMCs). Previously, CD5 depletion of PBMCs has been used to enrich for a CD5dim expressing subset prior to NK co-culture with an irradiated feeder line, but this can limit the yield of the final NK product. The purpose of this study was to compare ex vivo culture conditions using standard CD5 depletion versus unmanipulated PBMCs plus feeder line co-culture with 100 IU/mL rhIL-2 in matched healthy donors, hypothesizing that PBMCs plus feeder cells would generate an equivalent or superior NK product to CD5 depletion. Cell count, fold change, and viability data were collected for both techniques in 12 dogs across five time points up to day 14. A mixed-effects model analysis showed no statistical difference in calculated cell counts, overall fold change, and viability (p>0.05 all) between PBMCs with feeders and CD5 depleted cells with feeders. PBMCs had a higher mean than CD5 depleted cells at day 14 in all three categories, reaching a peak mean of 677 million cells from 5 million PBMCs at day 0. Flow phenotype showed similar upregulation of NKp46 and Granzyme B expression. Killing assays against melanoma and osteosarcoma targets demonstrated comparable results among PBMCs plus feeders versus CD5 depleted NK cells (p>0.05). Overall, these findings support the use of unmanipulated PBMCs plus feeder line coculture as an equivalent method to CD5 depletion in the expansion of canine NK cells for adoptive immunotherapy.

Supported by grants from National Institutes of Health/National Cancer Institute (U01 CA224166-01, 1R03CA252793, T32 CA251007-01)
Aggregation of IgM+, TCRβ+ and CD4+ Cells around melanomacrophage centers - evidence of an organized lymphoid structure in a teleost
Authors: Vaidyanathan Saraswathy P , Steinel Natalie C
Published: January 2023

The study of immunity in non-model organisms has the potential to reveal the evolved diversity of the immune system. However, such studies are hampered by the paucity of species-specific reagents. Advances in single-molecule RNA-ISH overcomes these setbacks and allows for the detection of potentially dozens of gene targets in a single tissue section. We examined the spatial expression of immune genes in the splenic tissue of a teleost, threespine stickleback, using the advanced multiplexed RNAScope Hiplex assay. Threespine stickleback, a small teleost found throughout the Northern Hemisphere, is an emerging immunologic model. It has been proposed that teleost humoral adaptive immunity initiates in the melanomacrophage center (MMC), a putative evolutionary precursor to germinal centers (GC), however this has not been directly tested. Teleosts lack GCs, yet still generate affinity matured antibodies in response to antigen challenge. We find that stickleback MMCs increase in size in response to antigen immunization but not to control injection, suggesting that MMC response is antigen driven, similar to the mammalian GC. Analysis of splenic tissue indicates that IgM mRNA expressing cells aggregate in discrete regions around stickleback MMCs. Similarly, CD4 and TCR-β expressing cells aggregate near IgM+ cells, comparable to the organization of B and T cells in mammalian GCs. These findings suggest that stickleback splenic tissues have an organized structure, focused around the MMCs, and support the hypothesis that MMCs could be the site of teleost adaptive immunity and the evolutionary precursors to GCs. This study also highlights the application of RNAScope in a non-model organism.

Supported by NIH RO1AI146168
Development of highly specific peptibody against Porcine epidemic diarrhea virus
Authors: Ryu Jihoon , Shin Hyun-Jin
Published: January 2023

Porcine epidemic diarrhea virus (PEDV) is an etiological agent of porcine epidemic diarrhea (PED), causing dehydration, vomiting, and acute watery diarrhea. Because it has a high mortality rate in neonatal piglets, PEDV is one of the critical pathogens affecting the swine industry. Since PEDV has shown rapid spreading in herds of swine, early diagnosis of this virus is very important to control the disease. Serological diagnosis test is a simple, rapid method for detecting PEDV infected pigs, and the development of PEDV-specific antibodies is essential for an accurate serological diagnostic test. Because conventional methods of development of monoclonal antibodies are time and hand-consuming work, recombinant antibodies are considered as an alternative strategy for the development of target-specific monoclonal antibodies. Peptibody is one of the promising recombinant antibodies that target-specific peptide is fused with the Fc domain of immunoglobulin G. The peptibody which have a specificity of peptides and immune function of Fc region as advantages have developed for therapeutic and diagnostic purpose. Here, we generated a PEDV-specific peptibody (PEDV-PB) that is fused with PEDV-specific peptide and mouse IgG2b Fc region. PEDV-specific peptide sequences were discovered by random phage display library and PEDV-PB was produced from Chinese hamster ovary (CHO) cells. PEDV-PB showed highly specific binding in several serological tests for PEDV, such as western blotting, immunofluorescence assay, and enzyme-linked immunosorbent assay. Our results suggest that PEDV-PB is very useful for the development of a rapid detection kit for PEDV.
Computational fingerprinting of immune-mediated pressure on SARS-CoV-2 viral evolution reveals preliminary evidence for immune-evasion
Authors: Hertz Tomer , Cohen-Lavi Liel , Sachren Sinai , Koren Eilay , Burkovitz Anat
Published: January 2023

The global emergence of SARS-CoV-2 variants and Omicron in particular, has highlighted the risk of novel variants that will evade adaptive immune responses following vaccination and natural infection. Determining which viral mutations escape T-cells and antibodies is crucial for designing therapeutics and vaccines and assessing the implications of newly emerging variants.

We developed an automated immunoinformatic pipeline that considers both data on viral sequences and experimentally identified T-cell and antibody responses, to identify specific viral mutations that may be associated with immune escape. We used an unsupervised clustering method to cluster a set of 108 non-redundant anti-SARS-CoV-2 spike protein antibodies with solved 3D structures from the PDB database, and identified 9 unique antibody clusters. We computed the predicted changes in binding energies (ΔΔG) for each antibody and each specific mutation within its contact footprint. Using these ΔΔG scores, we computed an antibody escape score for each mutation. We also predicted the change in binding between predicted and experimentally verified T-cell epitopes and their presenting MHC molecules. We ranked frequent spike protein mutations based on their predicted effect on immune evasion, as well as individual SARS-CoV-2 variants, with a specific focus on VOCs. Our novel immune escape scores identified several key mutations in the Omicron variant, which were not present in previous VOCs, and may be used to identify key mutations that are likely associated with immune escape, which may appear in novel variants of concern.

Supported by an NIH grant (75N93021C00016)
Identifying distinct T cell subsets in the context of pediatric ARDS
Authors: Clark Brandi L , Flerlage Tim , Schattgen Stefan A , Allen E Kaitlynn , Boyd David F , Crawford Jeremy C , Thomas Paul G
Published: January 2023

Acute respiratory distress syndrome (ARDS) is a condition defined by sudden respiratory failure caused by severe lung injury, with disease etiology that can differ across pediatric and adult patients. While T lymphocytes have been implicated in ARDS, the role of T cell subsets in pediatric ARDS (pARDS) is not fully understood. In this study, we sought to characterize the potential role of T cells in pARDS caused by viral lower respiratory tract infection (LRTI). To do this, we performed single cell RNA-Seq on tracheal aspirate samples from three categories of mechanically ventilated pediatric patients: 1) LRTI patients with pARDS, 2) LRTI patients without pARDS, and 3) ventilated patients without underlying LRTI or pARDS. All LRTI patients were diagnosed with respiratory viral infections, 70 percent of which were respiratory syncytial virus (RSV). Within the T lymphocytes, we identified populations of CD8+ T cells, CD4+ regulatory T cells, and γδ T cells that each expressed unique gene signatures with distinct distribution across disease states. We compared these data to T cell subsets within healthy lung tissue at multiple ages, and within the periphery of healthy adults. These analyses identified age, location, and disease-specific differences within T cell subsets, highlighting the unique role of T cells in pARDS.

Supported by grants from NIH (R01 AI54470, R01 AI121832)
DEC-205-expressing dendritic cells inhibit T cell responses during West Nile Virus encephalitis in the central nervous system
Authors: Mo Chiefe , Durrant Douglas Matthew
Published: January 2023

West Nile Virus (WNV), a neurotropic flavivirus, is the leading cause of mosquito-borne neuroinvasive disease in the U.S. Recent studies show that dysfunctional T cell responses correlate with severe disease in both humans and mice. Dendritic cells (DCs) are critical for reactivating antiviral CD8+ T cell responses in the CNS that are required to mitigate viral encephalitis. Previous reports have shown that CNS-localized DEC-205+ DCs accumulate within the CNS during WNV encephalitis; however, their specific role in programming T cell immunity remains poorly understood. DEC-205, an endocytic receptor expressed by DC subsets can promote either antigen-specific effector T cell activation or immunological T cell tolerance. Using a mouse model of WNV encephalitis, we demonstrate that DEC-205−/− mice exhibit enhanced clinical signs of disease and increased mortality compared to WT controls. The enhanced susceptibility in DEC-205−/− mice arose from an increased viral burden with the CNS; however, this was associated with increased inflammatory myeloid, CD4+, and CD8+ T cell infiltrates. In addition, expression of activation markers CD25 and CD44 increased while CD69 levels decreased within the DEC-205−/− mice compared with their WT counterparts. CD69 expression has been shown to be associated with regulatory T cells. To confirm the immunomodulatory role of DEC-205, we will measure CD4+ regulatory T cell frequencies within the CNS of DEC-205−/− and WT animals following WNV infection. Taken together, these data suggest that DEC-205 critically regulates WNV pathogenesis and neuroinvasive disease by limiting immune-mediated pathology in the CNS following WNV infection.

Supported by grants from NIH (SC3GM130472).
A Unique Cooperativity between Optineurin and CCL5 Triggers Early Cell Death during Herpes Simplex Virus-1 Infection of the Cornea
Authors: Patil Chandrashekhar Devidas , Shukla Deepak
Published: January 2023

Induction of cell death is considered an effective strategy to control viral infections. Rapid killing of infected cells prevents propagation of viruses and thereby, effectively reduces the infection. Herpes Simplex Virus-1 (HSV-1) is known to cause ocular and nervous system infections. While studying an autophagy receptor protein, Optineurin (OPTN), we found evidence of a unique collaboration between OPTN and CCL5 to generate signal for early cell death in HSV-1 infected cells in the cornea. OPTN knockout mice tend to die due to rapid spread of HSV-1 infection to the brain causing encephalitis. Interestingly, the OPTN knockout animals also exhibit a marked reduction in CCL5 induction. Human transcriptomic profile dataset shows a strong positive correlation between CCL5 upregulation and OPTN expression during HSV infection. Our data shows that a well-coordinated activity of both, OPTN and CCL5, is needed to trigger an early cell death signal that mimics pyroptosis to prevent viral spread. Downregulation of CCL5 and/or OPTN causes interference with this signal and a corresponding delay in cell death. Our results provide new information on an anti-HSV1 chemokine signaling pathway that participates in innate immune control of HSV-1 spread and viral transmission.
Influenza A virus-specific maternal antibodies impair vaccine-induced antibody responses and protection in male to a greater extent than female offspring.
Authors: Campbell Ariana Denise , Creisher Patrick S , Perry Jamie Lynn , Roznik Katerina , Sherer Morgan L , Burd Irina , Klein Sabra L
Published: January 2023

Influenza A virus (IAV) is a public health threat to pregnant people and infants. Transfer of antibodies across the placenta to the fetus and during lactation provides key protection from infectious pathogens during the neonatal period. Despite this, pathogen-specific maternal antibodies may interfere with infant vaccination. We hypothesized that transfer of IAV-specific maternal antibody, would negatively affect vaccine-induced immunity and protection in male and female offspring, with males being more impacted. Pregnant CD1 mice were infected on embryonic day 10 with 103 TCID50 of mouse adapted A/California/09 H1N1 (ma2009 H1N1) or mock inoculated. In offspring of IAV-infected dams, maternal anti-ma2009 H1N1 IgG peaked at 3-weeks, declined at 7-weeks, and undetectable at 15-weeks of age. At 3-, 7-, and 15-weeks, male and female offspring were intramuscularly vaccinated with inactivated ma2009 H1N1 vaccine, boosted 21 days later, and had serum collected 14 days post-boost. Following vaccination, 3-week-old offspring of IAV-infected dams displayed significantly lower vaccine-induced anti-ma2009 H1N1 IgG titers compared to offspring born from mock-infected dams. At 7 weeks of age, vaccine-induced IgG titers were significantly inhibited in male but not female offspring. In 15-week-old offspring of IAV-infected dams, vaccine-induced antibody responses were similar to offspring of mock-infected dams, being greater in female than male offspring. These data suggest that IAV-specific maternal antibodies negatively impact the offspring response to IAV-vaccination, with this more pronounced in males than females. This research is important for understanding the factors that impact vaccine-induced immunity in early life.

Supported by funding from the Johns Hopkins Vivien Thomas Scholars Initiative and grants from NIH/NICHD R01 HD097608, JH-CEIRS contract HHSN272201400007C, and NIH/NIA U54AG062333, NIH/NIAD 2T32A1007417-26
B cell-derived acetylcholine controls local inflammation and replication of influenza virus during respiratory tract infections
Authors: Prieto Antonio Cembellin , Murray Kaitlin , Reardon Colin , Baumgarth Nicole
Published: January 2023

Acetylcholine (ACh), a well-known neurotransmitter, is produced by the action of choline acetyltransferase (ChAT). Recently, ChAT expression was shown also in leukocytes, however, its function there remains to be more fully explored. Flow cytometry revealed that most ChAT-expressing cells in spleen, lymph nodes, lung, and pleural cavity of ChAT-GFP reporter mice are B cells, with B-1 cells having the highest frequency of ChAT+ cells among them. B cell-specific mb1-Cre ChATflx/flx knockouts (BChat-KO) had apparent normal bone marrow B cell development. Also, serum IgG and IgM levels and spontaneous IgM secretion in the spleen were comparable to wild-type littermate controls. Nonetheless, their spleen B-1 cell numbers were reduced. Intranasal infection of BChat-KO with influenza A/Puerto Rico/8/34 showed significantly smaller increases in IgM-secreting cells in the spleen and mediastinal lymph nodes (medLN) at 7 days post-infection (dpi) compared to controls. Inflammation-induced emigration of CD5+ B-1 cells from the pleural cavity to the medLN, which we showed previously to occur early during influenza infection, appeared exaggerated in the BChat-KO mice, leading to reductions in pleural cavity B-1 cells. Additional alterations to the macrophage and neutrophil composition suggested enhanced pleural inflammation in BChat-KO. This was correlated with reduced control of lung virus replication at 7 dpi. Overall, the data suggest a non-redundant function for B cell-derived ACh in the regulation of pleural inflammation and the control of virus replication following infection with influenza A.

The study was supported by a grant from the Chan Zuckerberg Initiative to Colin Reardon and Nicole Baumgarth
Impact of B-cell depletion therapy on respiratory viral infection
Authors: Allushi Bujana , Chlebicz Magdalena , Turner Sean , Kovats Susan , Axtell Robert C
Published: January 2023

B cell depletion therapy with anti-CD20 antibodies ameliorates autoimmune diseases such as multiple sclerosis, rheumatoid arthritis as well as haematological malignancies. Anti-CD20 antibodies deplete pre-B cells, mature B cells and malignant B cells but do not reduce plasma cells. Recently, studies have associated anti-CD20 therapy with severe manifestation of SARS-CoV2 infection. However, the mechanism of this adverse effect of anti-CD20 therapy remains unclear.

This study was undertaken to investigate the effect of anti-CD20 therapy on respiratory viral infection. C57BL/6 mice were treated with anti-CD20 antibody to induce B cell depletion and then were infected intratracheally with H1N1 Influenza A mouse-adapted A/Puerto Rico/8/1934 virus (IAV). Our results show that B cell depletion therapy worsens lung infection with more severe and prolonged weight loss. Higher viral titers were found in the lungs of anti-CD20 treated mice, indicating impairment in virus clearance. Flow cytometry analysis showed no difference between groups in CD4+ and CD8+ PR8 antigen specific T cells on day 9 post-infection. However, higher numbers of inflammatory, SiglecF+ neutrophils were found in B cell depleted mice.

In summary, our data show that anti-CD20 therapy exacerbates the viral pathology in respiratory lung infections. We further have identified that B cell depletion therapy affects viral clearance and inflammatory neutrophils but has no effect on T cell numbers in the lung at the peak of infection. Our future studies will identify the mechanisms driving the severe pathology in influenza infection with B cell depletion.

Supported by grants from NIH (R01AI137047 and R01EY027346) A grant awarded to Dr. Kovatz and Dr Axtell from the Presbyterian Health Foundation
Non-classical major histocompatibility factor H2-O counteracts gammaherpesvirus manipulation of the germinal center response
Authors: Stoltz Kyle , Golovkina Tatyana , Tarakanova Vera
Published: January 2023

Gammaherpesviruses, such as Epstein Barr virus, infect 95% of adults and form lifelong infection primarily through manipulation of B cells. During primary infection, gammaherpesviruses establish latent infections within B cells and drive robust, polyclonal germinal center (GC) responses, which atypically include B cells specific to both viral and self antigens. Consequently, this humoral response results in production of both autoantibodies, and interestingly, antibodies reactive to antigens of non-human species. While this non-physiological production of antibodies suggests viral manipulation of immune tolerance, the underlying mechanisms driving these phenomena are poorly understood.

Utilizing murine gammaherpesvirus 68 (MHV68), a rodent pathogen highly similar to EBV which serves as an animal model of EBV infection, we have discovered that the host factor H2-O (HLA-DO in humans) attenuates MHV68 latency establishment and the MHV68 driven GC response. H2-O acts as an inhibitor of H2-M (HLA-DM), the protein which regulates peptide exchange and thus the repertoire of peptides associated with the MHC-II peptide binding groove. We found that mice deficient in H2-O displayed increased MHV68 latent reservoirs, increased GC response, and a selective increase in dsDNA specific IgG, with no change in virus specific IgG at the peak of latent viral infection. These results show a novel role of H2-O in attenuating the non-physiological, self-directed B cell response driven by MHV68. Furthermore, these studies are the first to demonstrate that fine tuning of MHC-II antigen presentation is sufficient to alter the self-directed B cell differentiation usurped by a ubiquitous viral pathogen during establishment of life-long infection.
Modeling chronic gammaherpesvirus infection as a risk factor for MS reveals a profound impact of host genotype on control of viral load and T helper reprogramming
Authors: Holt Emily A , Lahue Karolyn G , Usherwood Edward J , Teuscher Cory , Krementsov Dimitry N
Published: January 2023

Multiple Sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) that impacts approximately 2 million people worldwide. The etiology of the disease is multifactorial, consisting of multiple environmental and genetic risk factors. A major risk factor is chronic infection with the gammaherpesvirus, Epstein Barr Virus (EBV). The mechanisms by which EBV increases MS risk, including the role of host genetics, are still unclear. To examine the role of host genetics on gammaherpesvirus infection outcomes, we infected male and female C57BL/6 (B6) and wild-derived PWD/PhJ (PWD) mice with MHV-68, a gammaherpesvirus homologous to EBV. Given the central role of CD4 T cells in MS, we first determined the effect of latent gammaherpesvirus infection on the CD4 T cell transcriptome. CD4 T helper cells were isolated from chronically infected mice followed by transcriptional profiling. Chronic MHV-68 infection resulted in a dramatic upregulation of genes characteristic of so-called cytotoxic T helper cell (ThCTL) phenotypes, including Gzmb, Cx3cr1, Klrg1, Prdm1, and Tbx21, which was most pronounced in B6 females, and highly muted in PWD mice of either sex. Flow cytometric analyses confirmed the genotype-specific expansion of ThCTL-like cells, beginning within 9 days of infection and continued into latency. Analysis of MHV-68 replication kinetics demonstrated that PWD mice had remarkably superior control of viral load early and late in infection. Because poor control of EBV viral load and enhanced anti-EBV responses are associated with greater MS risk, taken together, our findings suggest that the increased risk of MS due to EBV infection might be due to genetically determined poor control of viral load and augmented ThCTL responses.

Supported by a grant from the NIH (R21 NS095007)
IRF5 risk variants contribute to pre-symptomatic SLE by enhancing the levels of circulating NET antigens
Authors: Matta Bharati , Battaglia Jenna , Thomas Lydia , Barnes Betsy J
Published: January 2023

SLE is a complex multifactorial autoimmune disease characterized by high levels of autoantibodies that impact many organs. Neutrophil extracellular traps (NETs) are a potential source of antigen leading to the production of autoantibodies, as antibodies against NET components are detected in SLE patients. Higher level of NETs and decreased clearance of NETs are associated with SLE and other autoimmune diseases. Neutrophils play an key role in pediatric lupus and NETs from pediatric lupus patients activate plasmacytoid dendritic cells (pDCs) to produce high levels of IFN-α. Polymorphisms within and around the IRF5 gene associate with risk of developing SLE. We previously reported that healthy donor neutrophils from IRF5 homozygous risk carriers underwent increased spontaneous NETosis, as compared to non-risk donors, that resulted in increased IFN-α production, plasma cell differentiation and autoantibody production. Whether the observed increase in neutrophil-mediated ETosis is a systemic signature of IRF5-driven pre-symptomatic SLE is not known, nor is it known whether these IRF5 risk carriers have defects in NET clearance. We thus developed and optimized new assays to detect small quantities of NET remnants in plasma samples using a combined MPO-CitH3 antibody cocktail followed by detection of DNA by ELISA. Further, we designed a novel immunofluorescence technique to visualize and quantify plasma NETs in healthy donor risk and non-risk carriers, as compared to pediatric and adult SLE patients. In addition, plasma DNase 1 and DNase 1L3 levels were measured. This study will report on the mechanisms by which circulating NETs are increased in pediatric and adult SLE, as well as the contribution of IRF5 genetic risk to NET clearance.

Supported by Lupus Research Alliance Department of Defense CDMRP LRP W81XWH-18-1-0674
Androgens can modulate immunoregulation directly through gut cells and indirectly through alteration of microbiota composition/function in a lupus mouse model.
Authors: Ma Jing , Harder James , Alard Pascale , Kosiewicz Michele M
Published: January 2023

Hormones and gut microbiota affect lupus. In female NZBxNZW1(BWF1) mice, androgen treatment abrogates lupus; castration/treatment with androgen receptor blocker (flutamide) induces disease in BWF1 male mice. Gut microbiota alterations have been reported in lupus. Our goal is to understand the androgen/gut microbiota relationship, and its affect on lupus. We found male castration decreases peripheral, not thymic, derived Tregs; this correlates with decreased gut CD103 dendritic cells (CD103DC) function (induce Treg conversion) and RALDH2 [required for retinoic acid (RA) production) expression. CD103DC have very low androgen receptor (AR) expression, thus unlikely to be affected by androgens. We found that lamina propria stromal cells (LPSC), a major source of RA (via RALDH1/2 expression) required for normal gut CD103DC development, express very high AR levels. Castration/flutamide treatment in male mice decreased RALDH1/2 expression in LPSC, suggesting androgens may affect CD103DC function through LPSC. Castration/flutamide treatment also altered gut microbiota composition. Interestingly, intact male microbiota transfer to castrated male mice restored RALDH2 expression and CD103DC function, suggesting male microbiota factors may also alter CD103DC function. Metabolomic analysis of castrated male feces showed reduced phytanic acid (PA) levels. Administration of PA in vitro/in vivo restored castrated CD103DC function. These data strongly suggest that androgens alter (1) immunoregulatory function through gut cells and (2) gut microbiota composition/function that in turn modulate immunoregulatory function, and reveal how androgens may influence lupus and provide the basis for effective therapy development.

Funded by a TIL grant from the Alliance for Lupus Research and the NIH (R01AR067188)
Microbiome methods in experimental autoimmune encephalomyelitis.
Authors: Hoffman Kristina R , Daberkow David P , Kohl Hannah M , Long Tyrel , Kirby Trevor O , Ochoa-Reparaz Javier
Published: January 2023

Multiple Sclerosis (MS) is an autoimmune disease that affects the central nervous system (CNS) via neuroinflammation and demyelination. The exact triggers, subsets and effector mechanisms that contribute to disease progression are still largely unknown. Recent studies of healthy vs MS human stool samples indicated an altered microbiome, dysbiosis, which could lead to inflammation and disease. Experimental autoimmune encephalomyelitis (EAE) is a model used for the study of MS and can be induced in multiple non-rodent and rodent species. It is critical to control the environment of both the animal facility and experimental housing conditions in microbiome studies. We compared commercial vendors, Envigo and Jackson Laboratory, C57BL/6 female mice. Fecal samples were collected at Day 0, 14, and 21 for DNA extraction and sequencing of the ribosomal DNA (rDNA) to analyze the gut microbiome composition prior to and after induction of EAE. There was a significant difference between sources with Jackson Laboratory mice having an increased severity index compared to Envigo mice (p < 0.01) and a decreased survival rate of 20% when compared to 85% for Envigo mice. Our results suggest different sources of EAE mouse models have critical impacts on microbiome composition and levels of disease severity. Furthermore, this highlights the importance of consistent and controlled conditions from the animal model source, and throughout the experiment, when inducing EAE in mice and other animal models of disease.

This project was sponsored by NIH grant R15 NS107743.
T cell intrinsic role for NOD2 in Blau Syndrome
Authors: Napier Ruth J , Vance Emily E , Koney Kylie V , Lee Ellen J , Davey Michael M , Rosenzweig Holly L
Published: January 2023

Children with mutations in the nucleotide binding domain of NOD2 cause the monogenetic inflammatory disease Blau Syndrome, which is characterized by dermatitis, arthritis and uveitis. NOD2 is a well-known microbial sensor important in anti-bacterial immunity. However, the mechanism by which mutated NOD2 causes non-infectious inflammation in Blau is unknown. We recently reported a novel function of Nod2 as a central regulator of T cell homeostasis and as a negative regulator of autoreactive Th17 responses in experimental autoimmune uveitis (EAU) and arthritis. Here, we examined the effect of Blau mutations on T cell function and EAU using Blau knock-in mice carrying the most common Blau-mutation R314Q (corresponding to R334Q in humans) and Blau patient cells. We found that CD4+ T cells from Blau patients and Blau mice, produced increased IL-17 but decreased IFNγ following in vitro TCR-activation compared to control patients and WT mice, respectively. Blau mice developed worsened EAU compared to WT controls, which was accompanied by increased autoreactive (retina-specific) Th17 cells within the eye. Lymphocyte-deplete Rag1−/− mice reconstituted with Blau CD4+ T cells developed worse EAU than WT T cell recipients, indicating that CD4+ T cells expressing Blau-specific Nod2 mutations are sufficient to cause exacerbated EAU. Cumulatively, these data reveal a previously unconsidered role for Th17 cells in Blau Syndrome, and provide new T cell-targeted therapeutic avenues to treat this disease.

Supported by grants from VA (CDA-2 IK2BX004523 and Merit I01BX002180) and NIH (R01 EY025250).
Leveraging genetic correlation across immune-mediated disease to gain insights into underpowered studies of rare conditions
Authors: Molineros Julio , Elliott Katherine , Truong Dongnhu T , Fang Hai , Hart Amy , Li Shuwei , Waterworth Dawn , Knight Julian C , Black Mary Helen
Published: January 2023

Prevalence of immune mediated disease (IMD) within N. European populations varies from common (atopic dermatitis AD=10%) to rarer (giant cell arteritis-GCA=0.02%; hidradenitis suppurativa-HS=0.77%). This directly affects statistical power to detect genomewide SNP associations and to identify “causal” genes.

Objective

Leverage genetic correlations between differently powered IMD cohorts to study the genetic mechanisms underlying multiple immune-mediated diseases.

We used UK Biobank clinical data to create 18 IMD cohorts and a control set of individuals without any history of autoimmune disease (N=50,136). Using whole genome sequencing data from ~140K participants of the UKB, we performed SNP-level GWAS with REGENIE v2, and prioritized the associated genes using association statistics, in silico annotation, and eQTL information, as implemented in the Priority Index (Pi) algorithm. We used k-means clustering across relative gene rank distances optimized with the elbow method to group IMDs according to similar genetic architectures.

Result

IMD cohort size ranged from 196 to 3,411 cases and were each compared with a common set of controls. Optimal clustering yielded 4 groups, each with 6,5,4 or 3 IMD’s respectively. One cluster of interest contained HS, AD, vitiligo, GCA and Sjogren’s syndrome. This cluster reflects similarity in common “pathogenic” genes across IMD and highlights new candidate genes for further investigation that would otherwise go unnoticed in poorly powered studies of GCA and HS.

In conclusion, our proposed use of genetic associations and clustering of their relative ranks across multiple disease phenotypes identified pathogenic genes in understudied IMDs that would otherwise not have been elucidated.

Supported by Janssen Research & Development
Genes associated with Multiple Organ Dysfunction Syndrome during severe pediatric influenza
Authors: Novak Tanya , Crawford Jeremy Chase , Lange Christoph , Hahn Georg , Randolph Adrienne G
Published: January 2023

Influenza remains a global pathogen that causes significant morbidity and mortality even in previously healthy children. Multiple Organ Dysfunction Syndrome (MODS) is a potential complication developed during severe infection. This multi-site, national study investigated which genes are associated with resolving, developing, or predicting prolonged MODS in children admitted to ICU with severe influenza. PAXgene whole blood RNA from 216 influenza positive patients (median age 6.7 years, IQR: 2.6, 11.2) was extracted and hybridized to 469 NanoString nCounter® mRNA probes including positive/negative controls. Absolute mRNA molecules were quantified, normalized to 5 reference genes, and analyzed using linear models controlling for age, bacteria co-infections and type I error via FDR. Children who developed MODS had significantly higher levels of the antimicrobial Lactotransferrin (LTF) mRNA expression than those that never had MODS (p=0.04) whilst the latter group showed significantly higher expression of Transforming Growth Factor β1 (TGFBI) (p=0.04). In children with prolonged MODS, nine neutrophil degranulation genes were upregulated including TCN1, RETN, and LCN2 (p<0.001) which have previously been implicated in sepsis-related ARDS and mortality, and as biomarkers for severe influenza in adults. This study highlighted a panel of genes that are associated with MODS during severe pediatric influenza and could potentially be used for predicting and managing this complication.

Supported by grants from NIH/Department of Health and Human Services 1R21HD095228-01
Targeted transcriptomic signature for monitoring anti-tuberculosis treatment response
Authors: Manneh Ismaila L , Darboe Fatoumatta , Owolabi Olumuyiwa , Dockrell Hazel M , Sutherland Jayne S
Published: January 2023

The long duration of anti-tuberculosis treatment is associated with poor adherence and treatment outcomes leading to higher relapse rates. Shortening treatment duration has been suggested to improve treatment outcome by increasing adherence. Sputum conversion, the gold standard for monitoring treatment, has low sensitivity and specificity for predicting relapse. Better markers are therefore needed and blood-based biomarkers have been suggested as potential alternatives. The aim of this study was to identify transcriptional signatures to predict anti-tuberculosis treatment outcomes, relapse, and treatment failure. We extracted RNA from whole blood PAXgene samples from 37 confirmed TB cases at diagnosis (baseline), 14 days and two months of treatment and measured gene expression using targeted sequencing on the NanoString nCounter MAX platform using the 799-gene host response panel. To identify genes that predict treatment response and relapse, we identified differentially expressed genes (DEGs) at Day 14 and two months of treatment and compared expression between fast (sputum converters by 2 months) and slow responders (sputum converters after 2 months). Using a cut-off of 1.5-fold change and a p value of 0.05, we observed 58 DEGs at Day 14 and 175 DEGs at two months relative to baseline. When we compared slow and fast responders, 14 genes were differentially expressed but these were not significant when adjusted for multiple comparisons. We have shown that DEGs can identify different stages of disease or treatment. Further analysis is ongoing to include other treatment timepoints (four months and six months) and importantly to include participants with known relapse.