bluuebunny/crossref_metadata_2025 huggingface dataset:
Large-batch evaluation of cell counter instrument-to-instrument consistency for cell therapy applications
Authors: Bell Jordan , Huang Yongyang , Chan Leo
Published: January 2023

Cell therapy has been hailed as a medical revolution, and its rise has only increased the importance of accuracy and precision in cell counting. Ensuring the identity, purity, and viability of cell therapy products is critical for increasing efficacy and avoiding potential harmful side effects. The burden of sample analysis often rests on fleets of automated cell counters, leading to the need for confidence that multiple cell counters will give similar results.

In this work we compare groups of two distinct cell counting instruments. The first type is a well-established single-sample counter (Cellometer K2), similar to those often found in R&D laboratories. The second type of instrument (Cellaca MX) is a high-throughput cell counter that is often more suitable for a manufacturing environment than its single-sample counterparts.

We test the consistency of these cell counters using both live cells and fluorescent beads. We use samples of Jurkat cells to simulate immunotherapy products, and prepare the beads in fixed reference samples to allow comparison of multiple instruments over extended periods of time. We use these beads to investigate agreement among 60 Cellometer K2 instruments and 30 Cellaca MX instruments, what we believe is the largest group of cell counting instruments to be involved in a single comparison study.

Our work illustrates experimental approaches suitable for the comparison of multiple cell counting methods. We measure consistency among the cell counters, both within groups of similar instruments and between instrument types. This consistency encourages optimism for “future-proof” assays that can survive the transition from R&D to production with minimal adjustment.
Characterization of a Novel Anti-inflammatory Biogenic Amine
Authors: Santambrogio Laura , Clement Cristina C
Published: January 2023

Tryptophan (Trp) is an essential amino acid whose catabolism generates a cascade of over fifty molecules in a cell-specific manner. Three major Trp metabolic pathways have been described: synthesis of serotonin, synthesis of melatonin and generation of kynurenines. In mammalian cells, 90% of Trp is processed through the kynurenine pathway and kynurenine metabolites are regarded as one of the most powerful mechanism for immune regulation. We recently reported a novel biogenic amine, 3HKA, generated through a still uncharacterized lateral pathway of Trp catabolism. This novel biogenic amine is generated by both professional (Dendritic Cells) and non-professional (Lymphatic Endothelial Cells) antigen presenting cells and, differently from all others Trp metabolites, display unique “in vivo” immunosuppressive capabilities. In vitro, 3-HKA exhibited an anti-inflammatory profile by inhibiting the IFN-g-mediated STAT1/NF-kB pathway in both mouse and human dendritic cells (DCs) with a consequent decrease in the release of pro-inflammatory chemokines and cytokines; most notably, TNFa, IL-6, and IL12p70. In vivo, 3-HKA exerted protective effects in an experimental mouse model of Psoriasis by decreasing skin thickness, erythema, scaling and fissuring, reducing TNFa, IL-1b, IFN-g, and IL-17 production, and inhibiting generation of effector CD8+ T cells. Similarly, in a mouse model of nephrotoxic nephritis, besides reducing inflammatory cytokines, 3-HKA improved proteinuria and serum urea nitrogen, overall ameliorating the immune-mediated glomerulonephritis and renal dysfunction. Overall we propose that this novel biological amine is a crucial component of Trp-mediated immune tolerance.
Renal Tubular Cell Ferroptosis: A New Player in Pathogenesis of Lupus Nephritis
Authors: Scindia Yogesh , Ali Abdel , Desai Dhruv , Mehrad Borna , Morel Laurence , Conrad Marcus , Clapp William , Abdelmegeed Ahmed
Published: January 2023

Background

New research indicates that the injury to the renal tubular epithelial cells is a better predictor of renal outcomes in lupus nephritis (LN) as compared to glomerular injury. Iron accumulates mostly in the renal tubular cells of LN patients and murine models of LN. Ferroptosis is an incompletely understood form of cell death involving iron mediated lipid peroxidation, but its role in LN is not well established.

Experiment

LN and control patient biopsies were stained for markers of ferroptosis. Kidneys of female (MRL/lpr) and male (NZW X BXSB) F1, lupus mice were analyzed for markers of ferroptosis. Human proximal tubular cells (PTEC) were treated with LN serum with or without Liporxstatin-2, a novel ferroptosis inhibitor.

Results

Compared to controls, LN patients expressed higher levels of ACSL4 and 4HNE, the ferroptosis core proteins in the renal tubules. Compared to non-nephritic mice, nephritic mice had significantly higher gene expression of Aifm2, Acsl4, and Gpx1 as well as protein levels of Acsl4. Nephritic mice had an impaired renal glutathione synthesis pathway. Glutathione is an essential positive regulator of glutathione peroxidase 4 (Gpx4: ferroptosis inhibitor) which resulted in lower protein expression of Gpx4. Collectively, LN kidneys displayed a ferroptosis signature and was associated with an increase in Ngal and Kim1, PTEC injury markers. LN patients’ serum induced PTEC ferroptosis and associated pathology were significantly reduced by Liproxstatin-2.

Conclusion

Our data identify occurrence ferroptosis in renal tubular cell which can contribute to the pathogenesis of LN. Liproxstatin-2 mitigates human LN serum induced PTEC pathology and holds promise as an adjunct therapy to alleviate LN severity.

Supported by grants from Vifor Pharma
The Role of LAG3 in Antibody Responses To Kidney Transplantation
Authors: Nicosia Michael , Fan Ran , Lee Juyeun , Gorbacheva Victoria , Beavers Ashley , Dvorina Nina , Baldwin William M , Fairchild Robert L , Min Booki , Valujskikh Anna
Published: January 2023

The role of lymphocyte activation gene-3 (LAG3) in T cell functions is well studied, however its role in humoral immune responses remains poorly characterized. The goal of this study was to test the role of recipient LAG3 in a mouse model of renal allograft rejectionNaive B6.LAG3−/− mice have elevated numbers of follicular and memory T cells, and plasma cells compared to WT mice, as well as increased frequencies of memory T cells reactive to H-2Dd, H-2Ds, H-2Dq and H-2Dk alloantigens. When kidneys were transplanted from C3H donors to B6 WT and LAG3−/− mice, all C3H kidney allografts survived for > 60d in WT recipients, whereas LAG3−/− recipients rapidly reject allografts and have elevated serum creatinine levels at d14 posttransplant. Graft histology at rejection revealed minimal T cell infiltration, diffuse C4d staining, atrophic peritubular capillaries, endothelial swelling and edema characteristic of antibody mediated rejection (AMR). Compared to WT, LAG3−/− recipients had elevated frequencies of anti-donor IFNγ producing T cells and increased levels of donor specific antibody (DSA) against MHC-I and MHC-II. Depletion of CD8 T cells in LAG3−/− recipients did not alter rejection kinetics, while B cell depletion significantly extended C3H kidney allograft survival, suggesting the predominant role of alloantibody rather than T cell mediated rejection in these mice. However, neither T nor B cell conditional knockout recipients rejected the allograft demonstrating LAG3 expression on both cell types is necessary to mediate rejection.

These findings demonstrate that LAG3 regulates both T and B cell functions in response to kidney allografts, and is an attractive therapeutic target for the prevention of AMR.

Supported by grants from NIH (P01AI087586-10)
PARP-inhibition with IFNg in the ovarian tumor microenvironment induces immunogenic cancer cell death for sustained anti-tumor immunity.
Authors: Kinjyo Ichiko , Adams Sarah F
Published: January 2023

Poly(ADP-ribose) polymerase (PARP) inhibitors have been a remarkable breakthrough in the treatment of ovarian cancers with BRCA1/2 mutations. We have previously reported that PARP-inhibition with immune checkpoint blockade extended survival of BRCA1-deficient ovarian cancer (ov ca) preclinical models. A significant increase in IFNg producing effector T cells in the tumor environment promoted tumor cytotoxicity in a caspase-independent manner.

In this study, we hypothesized that in the presence of IFNg, PARPi may induce immunogenic cell death (ICD) rather than apoptosis and promote immune-mediated tumor control. In vitro, PARPi+IFNg treatment induces hallmarks of ICD in BRCAmt ov ca; such as cell surface translocation of calreticulin, ATP and HMGB-1 release but not by PARPi or IFNg single treatment. Additionally, the PARPi+IFNg treated Ov ca cells induced maturation and activation of dendritic cells with enhanced antigen-presentation to stimulate anti-tumor T cells. Evidence of immunogenicity was confirmed by the benefit of vaccination with PARPi+IFNg treated Ov ca cells in vivo. The vaccination established the long-lasting protective anti-tumor immune responses to clear sequential tumor inoculations. T cell-depletion and adoptive transfer experiments showed the ICD-induced tumor immunity was surprisingly, both CD4 and CD8 T cell-mediated. Currently, we are addressing whether anti-tumor antibodies contribute to tumor control.

Our results identified a novel mechanism by which PARP-inhibition with IFNg induces ICD in the ovarian tumor environment. These effects can be leveraged to enhance tumor clearance in the combination with immune therapies to modulate TME by stimulating tumor-immune cycles.

Supported by grants from NCI (R37 CA229221)
Dendritic cells to prevent cancer: Immune responses against neoantigens after dendritic cell vaccination of Lynch Syndrome patients
Authors: Abidi Asima , Westdorp Harm , Gorris Mark A J , Scheijen Blanca , Boller Anna-Lena , Irusquieta Cristina Berganza , Hoogerbrugge Nicoline , Schreibelt Gerty , de Vries Jolanda I J M
Published: January 2023

Lynch syndrome (LS) is an autosomal dominantly inherited syndrome caused by monoallelic germline aberrations affecting a DNA mismatch repair (MMR) gene, which leads to the development of microsatellite instability. Individuals with LS are 60–80% more prone to colorectal cancer (CRC). MMR deficiency in tumor DNA causes shifts in the translational reading frame resulting in the production of altered peptides called neoantigens. In a phase I/II clinical trial we vaccinated 20 healthy LS mutation carriers and 3 LS patients with surgically resected CRC with monocyte-derived dendritic cells (DC) loaded with neoantigens of TGF-βRII and caspase-5, and CEA.

The objective was to analyze the presence and functionality of antigen specific CD8+ T-cells in peripheral blood and biopsies of post-treatment skin tests. Cell lines stably expressing the (neo)antigen were generated and used in calcein based killing assays with expanded (neo)antigen specific patient CD8+ T cells.

The vaccination induced antigen specific T cell responses in 20/23 patients. Multifunctional antigen specific T-cells producing multiple cytokines and high amounts of interferon-γ upon peptide stimulation were detectable in peripheral blood. Moreover patient T cells of 3/4 patients tested demonstrated cytotoxicity against tumor cells presenting TGFβRII neoantigen, the T cell receptor repertoires of which had shared motifs in the clonotypes between patients as identified by Illumina MiSeq sequencing.

Conclusion-DC vaccination with (neo)antigens is well tolerated in LS patients and induces functional (neo)antigen specific T cells capable of eliciting a cytotoxic immune response thus able to enhance antitumor immunity in a prophylactic and therapeutic setting.
MARCKS mediates the AXL/PD-L1 signaling axis and contributes to an immunosuppressive environment in lung cancer
Authors: Yoo Estelle Heerim , Li Ji-Min , Hsu Ssu-Wei , Chen Ching-Hsien
Published: January 2023

The advent of immunotherapy with immune checkpoint blockade (ICB) has led to significant advances in lung cancer management; unfortunately, many issues remain to be resolved, especially regarding a low objective response rate. Through integrated large-scale omics data and biomarkers from published immune ICB trials, we identified the PIP2-binding protein, myristoylated alanine-rich C-kinase substrate (MARCKS), and the AXL receptor as top-ranked therapeutic targets in mediating immunotherapy resistance. Both immunofluorescence and immunoprecipitation data demonstrated that MARCKS forms a molecular complex with AXL in aggressive lung cancer cells. In a screening of 200 patients using immunohistochemistry staining, we observed a positive correlation between AXL phosphorylation and MARCKS expression in advanced lung cancer. MARCKS knockdown abated the activity of AXL and its downstream pathways including PI3K/AKT and STAT3/Src signaling. Data from our cytokine arrays showed upregulation of anti-tumoral cytokines including GM-CSF and CXCL10 in MARCKS-knockout cells. We further discovered that MARCKS acts in accordance with PD-L1 to modulate T cell activity and killing. Upon co-culture with MARCKS-expressing lung cancer cells, cytotoxic T cells expressed higher PD-1 levels on cell surfaces, whereas T cell killing activity was potentiated in the MARCKS-knockout group. In mouse xenograft models, mice bearing MARCKS-deficient tumors displayed increased sensitivity to PD-L1 treatment. Our data suggest an oncogenic role of the AXL-MARCKS axis in modulating the tumor immune microenvironment. Therapeutic targeting of MARCKS may warm up an immunologically “cold” microenvironment, sensitizing tumors to immunotherapy.

This work was supported by grants funded by the Department of Defense Kidney Cancer Research Program (KCRP) W81XWH1910831 (log# KC180170) and the California UCOP Tobacco-Related Disease Research Program (TRDRP) T29IR0704.
In situ analysis of the metastatic immune milieu in breast cancer brain metastasis at the single-cell level.
Authors: Longworth Aaron J , Evans Katrina , Lawson Devon
Published: January 2023

Despite advances in early detection and treatment, breast cancer remains the second-leading cause of death among women. Among these cases, incidence of breast cancer brain metastasis (BCBM) carries a poor prognosis with a mean survival of 6 months following diagnosis, demonstrating a drastic unmet need for effective treatments. As the most abundant resident immune cell in the brain, microglia are poised to be effective first-responders to combat tumor infiltration, yet their response to metastases has not been fully characterized. Prevailing literature suggests that microglia promote metastatic outgrowth, however these works fail to adequately discriminate microglia from other brain resident and infiltrating macrophages. Employing a mouse model harboring a deletion in a super-enhancer of macrophage colony-stimulating factor 1 receptor which results in a near-complete loss of microglia, we find more tumor outgrowth compared to animals with microglia. We subsequently compared the metastatic immune milieu in the microglia replete and deficient mice using single-cell RNA sequencing and Akoya’s CODEX (CO-Detection by indEXing) high-parameter immunofluorescence multiplexing platform. We demonstrate that microglia undergo a pro-inflammatory shift in response to BCBM and infiltrating lymphocytes fail to express the inducible costimulatory molecule, ICOS, in the absence of microglia. To understand how microglia promote productive T cell responses to BCBM, we used CODEX to interrogate 20+ different biomarkers in BCBM with or without microglia. Through spatial phenotyping of immune cells from mice with and without microglia we uncover the importance of the microglia/lymphocyte crosstalk in controlling BCBM outgrowth.

Supported by NIH Grant (R01 CA237376-02)
Spatial profiling of immune cell markers in FFPE tumor tissues using the RNAscope&[trade] HiPlex v2 in situ hybridization assay
Authors: Dikshit Anushka , Basak Sayantani , Chang Ching-Wei , Collins Kim , Bunting Michaeline
Published: January 2023

The tumor microenvironment (TME) is highly complex, comprised of tumor cells, immune cells, stromal cells, and extracellular matrix. This study demonstrates a highly sensitive and specific multiplexed technique, the RNAscope HiPlex v2 in situ hybridization (ISH) assay for spatial and transcriptomic profiling of target genes to assess immune regulation in FFPE tumor tissues.

The RNAscope HiPlex v2 assay iteratively multiplexes up to 12 targets in frozen and FFPE tissues. The novel FFPE reagent effectively reduces background autofluorescence, improving the signal to noise ratio. Here we investigate spatial expression of 12 immuno-oncology target genes, including tumor markers, immune checkpoint markers, immunosuppression markers, immune cell markers and secreted chemokines The targets were simultaneously registered using HiPlex image registration software v2 that enables background subtraction.

We visualized T cell infiltration and activation within tumors using CD3, CD8 and IFNG expression. Subsets of pro- and anti-inflammatory macrophages were detected by CD68 and CD163 expression Hypoxia markers HIF1A and VEGF indicated immunosuppressive state of tumor cells. Preliminary analysis and quantification of the HIF1A expression using HALO® image analysis showed higher copy numbers in the lung tumor as compared to the other tumors. We additionally showed the differential expression of immune checkpoint markers PDCD1, and CD274 within the TME.

Using a highly sensitive multiplexed RNAscope HiPlex v2 ISH assay, 12 key targets were spatially resolved in four different tumor types. The assay can provide valuable understanding of the biological crosstalk among various cell types in complex and heterogeneous tissues.
Peripheral CD4 T cell resistance to type I interferon defines outcome of PD1 blockade therapy in human cancer
Authors: Boukhaled Giselle M , Gadalla Ramy , Elsaesser Heidi J , Sacher Adrian , Butler Marcus O , Brooks David G
Published: January 2023

Type I interferons (IFN-Is) are paradoxically associated with both the success and failure of immune checkpoint blockade (ICB), with increased inflammation in the tumor associated with better response. To understand how IFN-Is modulate the immune response to ICB, we developed a mass cytometry approach to quantify pro- and anti-inflammatory features of IFN-I responsiveness at the single cell level. Using high dimensional analysis we show that the inflammatory responses that are beneficial in the peripheral blood are the opposite of what is considered beneficial in the tumor, thus identifying a functional disconnect between the peripheral and tumor immune compartments. We demonstrate that an initial resistance to IFN-I by CD4 T effector (Teff) cells in the peripheral blood is strongly associated to long-term benefit of anti-PD1 therapy in patients with melanoma, head and neck and lung cancers. By contrast, a strong IFN-I response correlates with progression and low survival probability. Upregulation of PDL1 by IFN-I did not relate to response, however patients with myeloid cells that initially reacted to IFN-I with higher IDO1 induction survived longer after anti-PD1. Single-cell RNA-sequencing stratified based on response to IFN-I identified transcriptional programs associated with therapy response, providing mechanistic insight into the peripheral cell states prior to therapy conducive to anti-PD1 success. Thus, contrary to a benefit of an initially inflamed tumor environment, an initially restrained CD4 Teff inflammatory response to IFN-I averts therapy failure and enables tumor control. This IFN-I response potential is a promising new biomarker for prediction of which patients will benefit most from anti-PD1 therapy.

Supported by Canadian Institutes of Health Research (CIHR) Foundation Grant FDN148386, the Canadian Cancer Society (CCSRI) Innovation Award No. 706230, the National Institutes of Health (NIH) grant AI085043, The Terry Fox New Frontiers Grant the Scotiabank Research Chair to D.G.B, the Princess Margaret Hold’em for Life Cancer Research Fellowship (G. M. B).
TCR-engineered neoantigen-specific CD4+ T cells mediate immunotherapy of a class II-negative murine squamous cell carcinoma
Authors: Brightman Spencer E , Becker Angelica , Thota Rukman , Naradikian Martin S , Griswold Ryan , Dolina Joseph S , Schoenberger Stephen P
Published: January 2023

The expansion of neoantigen (NeoAg)-specific T cells often accompanies clinical responses to immunotherapies such as immune checkpoint blockade (ICB), adoptive cellular therapy (ACT), and personalized cancer vaccines (PCV), highlighting their importance for antitumor immunity. While the functions of NeoAg-specific CD8+ T cells have been characterized, the role of NeoAg-specific CD4+ T cells is less well understood. To study the antitumor mechanisms of NeoAg-specific CD4+ T cells, we sorted single CD4+ T cells specific for a mutated clathrin heavy chain epitope (Cltc H129Q) expressed by the murine squamous cell carcinoma VII (SCC VII) tumor. T cell receptor (TCR) sequencing analysis revealed the presence of four distinct TCR clonotypes and expression of these TCRs in primary cells was sufficient to confer preferential recognition of Cltc H129Q as compared to the corresponding wildtype Cltc epitope. Despite differences in TCR avidity, both low and high avidity CD4+ T cells were capable of proliferating to similar degrees in response to tumor antigen in vivo and enhancing primary tumor immunity in a CD8+ T cell- and CD40L-dependent manner. ACT with Cltc H129Q specific CD4+ T cells also synergized with cyclophosphamide treatment to reduce disease burden in mice with established tumors. Overall, these findings illuminate the mechanistic role of NeoAg-specific CD4+ T cells in tumor immunity, provide insights into the impact of TCR avidity on the helper functions of CD4+ T cells, and highlight the therapeutic potential of ACT with TCR-engineered CD4+ T cells.
Assessing Immunogenicity of Vaccines and Adjuvant Combinations for Q Fever
Authors: Jan Sharon , Gregory Anthony , Felgner Jiin , Davies Jenny , Nakajima Rie , Jasinskas Algis , Davies David Huw , Felgner Philip L
Published: January 2023

There is an unmet need for developing a safer, effective, single-dose vaccine for Coxiella burnetii conferring prolonged resistance, due to its high infectivity and biothreat potential. C. burnetii causes Q fever, which acutely causes fever-like symptoms but may manifest chronically and escalate into pneumonia and endocarditis. Prior sensitization has been documented to exacerbate the current vaccine’s reactogenicity and there have been considerable efforts in developing alternatives. Our lab has been studying the effects of combinations of adjuvants and their synergistic effects in eliciting antibody and T cell responses. Here we describe an effective adjuvant combination using synthetic TLR4 agonist monophosphoryl lipid A (MPLA), TLR9 agonist CpG ODN 1018, and Addavax, a squalene oil-in-water emulsion. In vitro assays were performed to assess the immunogenicity of these adjuvants in the context of NFκB activation and inducible nitric oxide synthase (iNOS) production.

Model antigens from C. burnetii were selected based on previous protein microarray data and formulated with varying adjuvant combinations. These formulations were administered to C57BL/6 mice and assessed for immunogenicity. Sera collected at regular intervals were screened against protein microarrays to monitor the dynamics and isotypes of the antibody response. Here we observed robust, durable and antigen-specific T cell and antibody responses. Our top vaccine formulations were administered to Hartley guinea pigs in a challenge study. Initial data demonstrate specific IgG production against immunizing antigens. Downstream analysis for this data set is currently underway.

Supported by DTRA contract HDTRA1-16-C-0009 DTRA grants HDTRA1-18-1-0035 and HDTRA1-18-1-0036
The novel adjuvant, ADA-1, restores age-associated defects in the adaptive immune response to Clostridioides difficile infection and vaccination in an aging mouse model
Authors: Bell Matthew , Bernui Mariana , Shah Neev , Connors Jennifer R , Kutzler Michele A
Published: January 2023

Clostridioides difficile is the leading cause of healthcare-associated infection in the United States. The elderly (≥65) have the greatest risk of C. difficile infection (CDI), severe morbidity, and mortality due to CDI. Though antibody responses against C. difficile Toxin A/B (TcdA/B) following primary infection or vaccination are protective against symptomatic disease, the elderly have impaired humoral responses. Using an aging mouse model, we show that there is significantly greater morbidity and mortality in aged mice during primary CDI compared to young adult mice, as seen in elderly CDI patients. Additionally, during both primary CDI and vaccination against TcdA/B, aged mice have significantly lower anti-TcdA/B antibody titers. This impaired humoral response is the result of poor Tfh and B cell responses following infection and vaccination as aged mice have a significantly lower frequency of Tfh cells and number of anti-TcdA/B B cells compared to young adult mice. While these age-associated defects in the humoral response allow for increased vulnerability to CDI, we show that these defects can be restored with the addition of the novel adjuvant ADA-1 during vaccination of aged mice with TcdA/B-encoding DNA. With ADA-1, the anti-TcdA/B antibody response in aged mice is restored to levels seen in young adult mice. ADA-1 also boosts the frequency of Tfh cells in aged mice following vaccination, suggesting that ADA-1 can restore the development of the aged germinal center reaction following vaccination. As such, this work highlights both the need for the development of a C. difficile vaccine with the elderly in mind and presents ADA-1 as a solution to overcoming age-associated defects in the vaccine response of the elderly.

Supported by Department of Defense Grant: W81XWH0910382 Pennsylvania Department of Health CURE Grant
Examining effector functions of lung CD8+ tissue resident memory T cells in humans.
Authors: Mattingly Cameron , Elliott Jennifer L , Thomas Jeronay K , Lobby Jenna L , Michalets Sarah E , Kohlmeier Jacob E
Published: January 2023

Due to their position in the lung tissue, CD8+ tissue resident memory T cells (TRM) act as sentinels of the respiratory tract that rapidly respond to, and mediate protection against, respiratory viruses. In mice, TRM have been shown to mediate protection at barrier sites by producing cytokines, chemokines, and performing cell lysis. In the lungs specifically, our lab has shown that influenza-specific CD8+ TRM rapidly produce IFNγ, but airway TRM are poorly cytolytic in mice. In humans, less is known about the effector functions of virus-specific CD8+ TRM in the lungs, and thus this study seeks to fill that gap in knowledge. Using cells from healthy human lungs, we first identified and quantified the frequency of antigen-specific cells in our lung donors by performing intracellular cytokine staining (IFNγ+) and activation induced marker assays (CD137+ CD25+). Then, by performing a series of in vitro peptide stimulation and cytokine neutralization experiments, we investigated which cytokines are produced by lung CD8+ TRM, and how those cytokines impact local innate and epithelial cells. Initial results show that, when stimulated with their cognate antigen, lung CD8+ TRM produce cytokines that directly activate innate immune cells. Results of this study suggests that human CD8+ TRM in the lungs act to rapidly reprogram local immune cells, and these data will ultimately help us understand how CD8+ TRM fit into the overall immune response to respiratory viruses.

Supported by grants from the NIH/NHLBI (R35 HL150803) and by NIH/NIAD through the Centers of Excellence for Influenza Research and Response (75N93019R00028).
MicroRNA-29a attenuates exhaustion and promotes memory-like CD8 T cells
Authors: Leng Xuebing , Buchness Lance , Ristin Svetlana , Villarino Alejandro , Stelekati Erietta
Published: January 2023

CD8 T cells are crucial for immunity against pathogens and tumor cells. However, persistent antigens during chronic infections and cancers drive the differentiation of exhausted T cells (TEX). TEX do not generate optimal immune responses to persistent antigens. The epigenetic profile of TEX is altered and cannot be reversed with current immunotherapy, such as PD-L1 blockade, providing a challenge for the efficacy of such therapies. Despite considerable work interrogating the transcriptional regulation of TEX, the post-transcriptional control of TEX remains poorly understood. Here, we interrogated the role of microRNAs (miRs) in CD8 T cells responding to acutely resolved or chronic viral infection and identified miR-29a as a key regulator of TEX. Enforced expression of miR-29a improved CD8 T cell responses, attenuated exhaustion, and fostered a memory-like CD8 T cell differentiation state during chronic infection. Importantly, miR-29a combined with anti-PD-L1 therapy increased CD8 T cell accumulation, promoted a progenitor-like TEX subset and enhanced a memory-like phenotype long-term. These data indicate that miR-29a can instruct CD8 T cell differentiation and promote memory-like responses. Therefore, we suggest miR-29a as a novel immunotherapeutic to regulate TEX differentiation and promote functional CD8 T cell responses long-term.
Genetic analysis of CNS autoimmunity using the diversity of the Collaborative Cross reveals unique phenotypes and mechanisms
Authors: Holt Emily A , Lahue Karolyn G , Mahoney J Matthew , Teuscher Cory , Krementsov Dimitry N
Published: January 2023

Multiple Sclerosis (MS) is a complex disease with remarkable heterogeneity in disease course and progression, the genetic basis of which remains obscure. Here we leveraged the Collaborative Cross (CC) - a highly genetically diverse mouse strain panel - and myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55) induced experimental autoimmune encephalomyelitis (EAE), to model genetics of MS disease course. 33 CC strains were selected based on compatible MHC haplotypes (H2b and H2g7), which captured a wide spectrum of distinct EAE phenotypes, compared with typical chronic EAE in C57BL/6 mice. CC028 mice exhibited severe and rapidly progressing disease. In contrast, several strains, including CC011 and CC040, were highly resistant to EAE. Sex differences in EAE course were observed in 4 strains, including CC042. Remitting-relapsing EAE was observed in 4 strains, including CC002. In addition to classical EAE clinical signs (ascending paralysis), we identified two strains, CC004 and CC083 that exhibited high incidence of axial rotary (AR)-EAE, including profound ataxia, head tilt, and axial rotation. Preliminary quantitative trait locus (QTL) analysis revealed a distinct linkage pattern in each sex for classical EAE severity, with several emerging peaks on chromosome (Chr) 2, 8, 12, 18, and X in females, Chr10 in males, and Chr4 in both sexes. QTL analysis of AR-EAE severity revealed a narrow interval on Chr18 (39.2–41.0Mb) passing suggestive linkage significance at the 90% confidence threshold. Experiments are ongoing to determine the immunopathologic basis of distinct genetically controlled EAE phenotypes in CC strains of interest.

Supported by grants from NIH (R21 AI145306-01) and the National MS Society (RG-1901-33309)
Common gamma chain-dependent cytokines drive antigen-independent proliferation of circulating and resident memory CD8+ T cells
Authors: Jarjour Nicholas N , Wanhainen Kelsey M , Peng Changwei M , da Silva Henrique Borges , Martinez Ryan J , Dalzell Talia S , Huggins Matthew A , Urban Joseph F , Hamilton Hart Sara E , Jameson Stephen C
Published: January 2023

Memory CD8+ T cells are found throughout the body and can locally control reinfection at barrier sites. Vaccines often fail to elicit durable tissue-resident memory (TRM) CD8+ T cells, likely contributing to unsuccessful efforts to vaccinate against several important pathogens. TRMs locally self-renew by poorly understood mechanisms; a better understanding of this process could help to enhance TRM immunity. We propose that proliferating TRMs flexibly use available members of the common gamma chain-(γC)-dependent cytokine family to persist and as bystanders during other immune responses. We have found that γC-dependent cytokines can mediate cell-intrinsic signaling to drive proliferation of both circulating and resident memory CD8+ T cells, in contrast to naïve T cells. Using adoptive transfer as well as tetramer staining of endogenous populations, we have confirmed that cytokine-stimulated proliferation of memory T cells of known antigen specificities elicited by different pathogens can occur without cognate antigen. Via bulk and single-cell RNA-Seq of resting and in vivo cytokine-stimulated memory CD8+ T cells, we are elucidating the downstream signature of cytokine-driven TRM proliferation. We have also found that memory CD8+ T cells proliferate comparably in response to exogenous cytokines or unrelated pathogens, consistent with pathogen-elicited γC-dependent cytokine signals driving memory CD8+ T cell proliferation. These data support a conserved ability of TRM and circulating memory T cells to use infection-elicited γC-dependent cytokines for enhanced proliferation, likely to promote maintenance, and that treatment with γC-dependent cytokines can drive rapid proliferation without a need to identify antigens.

N. N. J. Damon Runyon Cancer Research Foundation Fellow DRG-2427-21 K. M. W. NCI F30 H. B. d. S. CRI/Paul Shiverick Fellow; NIAID K99/R00 AI139381 S. C. J. NIAID R01 AI038903
Transient Receptor Potential Ankyrin 1 (TRPA1) mediates IL-1β-Induced Thermoregulation
Authors: Silverman Harold A , Tynan Aisling , Chang Eric H , Li Jian Hua , Thompson Dane A , Tracey Kevin J , Chavan Sangeeta S
Published: January 2023

TRPA1 is a non-specific cation channel, that has been previously implicated in temperature regulation and inflammation. Administration of TRPA1 agonists induce significant changes in body temperature. Similarly, IL-1β, a cytokine mediator of inflammation and injury, induces a significant change in body temperature. Here we reasoned that TRPA1 plays a role in IL-1β mediated temperature regulation. To monitor body temperature in conscious and unrestrained mice, telemetry probes were implanted into the peritoneal cavity. Using chemogenetic technology, we generated a novel TRPA1-GqDREADD strain, in which TRPA1+ cells are activated by the administration of clozapine-N-oxide (CNO). Administration of CNO (ip) induces a significant change in body temperature (vehicle vs. CNO; 674.3 ± 29.3 AUC vs. 1646 ± 172 AUC; Welch’s T-test; p<.001). To determine if TRPA1 is responsible for IL-1β induced thermoregulation, wild type mice and TRPA1 KO mice were injected with IL-1β (5.0μg/kg, ip). As expected, wildtype mice had a significant change in body temperature. However, this response was significantly impaired in TRPA1 KO mice (wild type vs. TRPA1 KO; 1045 ± 35.29 AUC vs. 577.9 ± 26.13 AUC; Welch’s T-test; p<0.0001). To investigate whether selective TRPA1 expression in neurons is required for IL-1β-induced thermoregulation, we generated Syn-Cre/TRPA1fl/fl mice selectively devoid of TRPA1 expression in neurons. Selective ablation of TRPA1-expression in neurons significantly attenuates IL-1β-induced changes in body temperature (wild type vs. Syn-Cre/TRPA1fl/fl; 1082 ± 61.49 AUC vs. 775.8 ± 66.89 AUC Welch’s T-test; p<0.01). Together, these studies indicate that IL-1β-induced thermoregulation is mediated via neuronal TRPA1.

Supported by grant from NIH to KJT and SSC
T cell derived XCL1 facilitates the interaction with XCR1+ DCs within the CNS during WNV encephalitis
Authors: De La Torre Kristen , Durrant Douglas
Published: January 2023

West Nile Virus (WNV) is a mosquito-borne flavivirus that causes encephalitis, especially in elderly and immunocompromised individuals. Previous studies have demonstrated that CNS-localized dendritic cells (DCs) protect against WNV neuroinvasive disease by establishing antiviral T cell responses in the CNS. In this study, we investigated the role of the XCL1-XCR1 interaction since its primary function is to promote DC-mediated CD8+ T cell immunity. XCR1, a chemokine receptor expressed predominately by a subset of DCs, mediates their migration in response to its ligand XCL1. We found that XCR1-deficient (XCR1−/−) mice demonstrated increased symptomatic disease and mortality after subcutaneous infection with WNV. In addition, WNV-infected XCR1−/− mice exhibited significantly increased viral burden in the CNS at day 12 post-infection. Within the WT CNS, we found increased XCL1 expression on day 6 post-infection. In contrast, the expression of its receptor, XCR1, increased on day 9 post-infection compared with uninfected CNS. As XCL1 is known to be produced by NK and CD8+ T cells, we next sought to determine which cells express XCL1 and at which locations during WNV encephalitis. Interestingly, compared with splenic T cells, CD8+ T cells isolated from the CNS and cervical lymph nodes from WT mice showed high XCL1 expression. These observations lead us to hypothesize that T cell derived XCL1 facilitates interactions with XCR1+ DCs within the CNS during WNV encephalitis.

Supported by grant from NIH (SC3 GM130472-03)
IL-9 has an important role in promoting Barrett’s esophagus (BE) phenotype in eosinophilic esophagitis (EoE)
Authors: Venkateshaiah Sathisha Upparahalli , Sekhar Yadavalli Chandra , Lokanatha Oruganti , Mishra Anil
Published: January 2023

Objectives

Establish the role of IL-9 in development of Barrett’s esophagus (BE) in human EoE.

Methods

Histopathological, immunohistochemical, RT-PCR and ELISA analyses are performed to identify the signature genes and proteins involved in the progression of BE in EoE.

Results

We detected characteristic feature of BE like intermediate columnar type epithelial cells, induced BE signature genes like ErbB3, CDX1, ErbB2IP in the esophageal mucosa of EoE patients. Further, we observed BE associated diagnostic proteins (TFF3, p53) and the progression markers (EGFR, p16), MHC molecules (MICA, MICB) in esophageal biopsies of chronic EoE patients. Furthermore, we also detected mucin-producing columnar cells and MUC-2, MUC-4 and MUC5AC genes and proteins along with induced IL-9 in chronic EoE patients. Interestingly, a strong correlation of IL-9 with mucin genes were observed that indicate IL-9 may have an important role in BE development in chronic EoE patients. Herein, we present in vitro data of IL-9 exposure to primary esophageal epithelial cells provide evidence that IL-9 is involved in the induction of mucins producing and epithelial barrier cells transcripts and proteins like CK8/18, GATA4, SOX9, TFF1, MUC5AC, including tight junction proteins that are the markers of BE.

Conclusions

Taken together, we provide evidence that IL-9 has a role in the initiation and progression of BE characteristics like development of mucin producing columnar epithelial cells in chronic EoE patients.

Supported by grants from NIH (R01 AI080581-Anil Mishra)
Characterization of a new cytokine complex from the IL-6/IL-12 family
Authors: Rousseau Marine , Laplante Véronique , Nadeau Ulysse , Pasquin Sarah , Fajardo Ernesto , Lesage Sylvie , Gauchat Jean-François
Published: January 2023

Cardiotrophin-like cytokine factor 1 (CLCF1) is a neurotrophic factor initially identified as a B lymphocyte stimulating factor. Mutations in CLCF1 cause type 2 Cold-Induced Sweating Syndrome, a severe multisystem disorder coupled with recurrent childhood infections. The CLCF1-CRLF1 composite cytokine mediate its neurotrophic effects via the CNTF receptor (CNTFR). Although immune cells do not express CNTFR, immunological activities of CLCF1 have been described, suggesting the existence of an alternative receptor. Preliminary results suggest that CLCF1 can associate with the Epstein-Barr virus-induced gene 3(EBI3). EBI3 is a subunit of the composite cytokines IL-27, IL-35 and IL-39, which exhibits immunoregulatory functions. Our hypothesis is that CLCF1 associates with EBI3 to activate an alternative receptor expressed by immune cells. The aim of the project is to identify the cells producing CLCF1/EBI3, to define its receptor and to characterize the influence of CLCF1/EBI3 on lymphocyte proliferation, differentiation, and function. We find that EBI3 and CLCF1 subunits can associate extracellularly to form a CLCF1/EBI3 complex. Moreover, we uncovered that this complex can bind to IL-12Rß1, IL-23R and CNTFRα receptor chains. Altogether, our data strongly suggest that the CLCF1/EBI3 complex is likely to assemble in vivo and act as a cytokine through known receptor chains. Overall, defining the cellular targets and biological activities of CLCF1/EBI3 will pave the way for the development of treatments in autoimmune diseases involving CLCF1 and EBI3, adding to the increasing number of biologic drugs targeting cytokines, their receptors, or signaling pathways that are used clinically to treat inflammatory diseases.

Supported by IRSC (RNI00404)
HSC-independent definitive lymphopoiesis persists into adult life
Authors: Yoshimoto Momoko , Cheng Haizi , Nishida Chika , Latorre Augusto , Kobayashi Michihiro
Published: January 2023

The stem cell theory that all blood cells are derived from hematopoietic stem cell (HSC) is a central dogma in hematology. However, exceptions are the fetal hematopoiesis in which primitive and definitive type of erythromyeloid cells are already produced from hemogenic endothelial cells (HECs) before the first HSC emergence in the mouse embryo. This early blood cell production prior to HSC emergence is called HSC-independent hematopoiesis and considered to be transient during fetus. Recent lineage-tracing studies revealed that some tissue-resident innate immune cells (e.g. brain microglia, mast cells, and certain types of γδT cells) are also produced independently of HSCs from early extra-embryonic yolk-sac (YS). Definitive T&B-lymphoid potential and multi-potent progenitor (MPP) potential have been detected in the YS and/or embryos in in vitro studies, however, the physiological presence of these cells in the embryo at pre-HSC stage remains unknown. In addition, whether HSCs in the fetal liver are the main source of innate-like B-1a cells has been controversial. Here, using complementary lineage tracing mouse models, we show that HSC-independent MPPs and HSC-independent adoptive T & B-lymphocytes persist into adult life and are gradually replaced by HSC-derived cells. Furthermore, we found that HSCs minimally contribute to the peritoneal B-1a cells, and most B-1a cells are originated directly from ECs in the YS and embryo and HSC-independent for life. Our discovery of extensive HSC-independent MPP and adoptive lymphocytes in adults attests to the complex blood developmental dynamics through embryo to adult that underpin the immune system and challenges the paradigm of HSC theory in hematology.

Supported by grant from NIH R01AI121197.
Vhl deficiency in Dmp1-expressing cells affects myeloid development and erythropoiesis: possible effects on myeloerythroid metabolism
Authors: Chicana Betsabel J , Emery Janna , Taglinao Hanna , Donham Cristine , Manilay Jennifer
Published: January 2023

The specific types of bone marrow (BM) stromal cells and local BM microenvironments that contribute to the regulation of myelopoiesis are not fully understood. The von-Hippel Lindau protein (VHL) controls responses to hypoxia and could regulate immune metabolism. We investigated this in Dmp1-Cre; Vhl conditional knockout mice (cKO), where Vhl is deleted in mesenchymal stem cells, osteoblasts (OBs) and osteocytes. Similar to previous findings when Vhl is deleted in OBs, we observed low blood glucose levels in the cKO mice, which may reflect an increase in OB cellular glycolysis and increased glucose uptake in the bone. Erythropoietin (Epo) treatment in vivo reduced blood glucose levels in rats. In preliminary studies, we found elevated Epo levels in cKO peripheral blood serum and BM fluid by 6 weeks of age, and evidence for dysregulated erythropoiesis. Moreover, the cKO displayed an increased frequency of common myeloid progenitors, CD11b+ Gr1− monocytes, and CD11b+ Gr1+ granulocytes by 10 weeks of age. We hypothesize that alterations in skeletal glucose metabolism directly affect myeloerythroid development in the BM through increased Epo-receptor (EpoR) signaling. EpoR is expressed on osteoprogenitors, hematopoietic stem cells, and B cells, but whether it is expressed on myeloid progenitors is unclear. Studies are in progress to test the hypothesis that overproduction of Epo by Vhl-deficient Dmp1+ cells results in elevated EpoR signaling in myeloid progenitors and lineages; this in turn, results in an increase in myeloid cell glycolysis and Glut1 expression and proliferation. These studies are relevant for the understanding how BM microenvironmental changes can trigger adaptations in immunometabolism of myeloid cells.

Supported by grants from NIH (R15 AI154245-01, F31 AI154815), and University of California, Merced
Cellular and molecular mediators of thymic DC homeostasis and activation
Authors: Srinivasan Jayashree , Helm Bryan , Su Zhe , Yi Song , Liu Qi , Lau Ken , Ehrlich Lauren Ilyse Richie
Published: January 2023

Central tolerance in the thymus ensures that autoreactive thymocytes undergo negative selection or diversion to the regulatory T cell (Treg) lineage. Thymic antigen presenting cells (APCs) such as medullary thymic epithelial cells (mTECs) and dendritic cells (DCs) present myriad self-peptides to thymocytes to induce central tolerance. We and others have previously shown that thymic cDCs can be subdivided into activated CCR7+ MHC-IIhi and resting CCR7− MHC-IIlo cDC1 and cDC2 subsets with distinct functional properties, suggesting the thymic DC compartment may be more heterogeneous than previously described. Here, we performed single-cell RNA sequencing (scRNAseq) of hematopoietic APCs isolated from adult mouse thymi to comprehensively identify thymic DC subsets with distinct gene expression profiles. Our data reveal 11 transcriptionally distinct cDC subsets. We identify 6 cDC1 subsets, 3 cDC2 subsets, 2 activated CCR7+ “cDC3” subsets distinguished by expression of cDC1 and cDC2 markers, and 4 plasmacytoid DC clusters. Through functional assays, we find that the XCR1+ cDC3s are the most efficient at presenting mTEC-derived peptides on MHC-I and MHC-II, while 2 distinct cDC2 subsets are the most efficient at generating Tregs in vitro. Additional scRNA-seq and flow cytometry analyses of thymic DCs from mice in which thymocyte maturation was blocked at different developmental stages reveal candidate cellular and molecular crosstalk signals that regulate homeostasis and/or activation of pDC and cDC subsets. Our findings reveal hitherto unknown functional heterogeneity within the thymic DC compartment and identify potential interactions that support DC homeostasis and activation which in turn could impact central tolerance.

Supported by NIH P01 AI139449-02