bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantifying in vitro complement-dependent cytotoxicity (CDC) using advanced flow cytometry
Authors: McBain Kirsty A , Lovchik Julie , Bevan Nicola
Published: August 2023

Complement dependent cytotoxicity (CDC) is a key Fc mediated function of monoclonal antibody (mAb) therapeutics. mAb binding to antigen triggers a complex molecular cascade with sequential recruitment of serum proteins, eventuating in target cell lysis. Here we demonstrate CDC quantification in a streamlined, in vitro, advanced flow cytometry assay.

Anti-CD20 mAbs were incubated with target cell lines in 96- or 384-well plates. Human serum (15%) was then added to induce CDC. Cells were labeled with iQue® Cell Membrane Integrity (R/Red) Dye to enable assessment of cell death using the iQue® Advanced Flow Cytometry Platform.

Induction of CDC by anti-CD20 mAbs correlated with target cell CD20 expression, with most cell death observed with high-CD20 expressing Ramos cells. Maximal Ramos cell death induced by Rituximab and Truxima® was 68% and 70%, respectively. Comparatively, cell death of mid-CD20 expressing Raji cells was lower at 34% and 24%. No CDC was induced with CD20 negative Jurkat cells. Another anti-CD20-IgG1 mAb was compared against two isotype mutants: IgG1fut (non-fucosylated) and IgG1NQ (non-glycosylated). Their activity was profiled towards three previously described effector functions: antibody-dependent cellular cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP) and CDC. CDC induction was comparable between the three mAbs, however their ADCC and ADCP activity differed. The IgG1NQ did not exert ADCP or ADCC activity, whilst the IgG1fut showed more ADCC and less ADCP than the native.

These data exemplify the use of advanced flow cytometry to quantify Fc function of mAbs towards a range of effector mechanisms, highlighting the potential to profile libraries of novel therapeutics in minimal time.
Novel Strategy to Regulate PD-L1 Expression and Enhance Anti-Tumor Effect
Authors: Sredni Benjamin , Chaouat Abigael , Hay Ophir , Manoim Julia , Nadler Ronia , Kalechman Yona
Published: August 2023

The programmed death 1/PD ligand 1(PD-1/PD-L1 axis is a major player in the tumor immune evasion strategy. We show that the immunomodulatory small tellurium molecules, AS101 [ammonium trichloro(dioxoethylene-o, o′)tellurate] and SAS [octa-O-bis-(R,R)-tartarate ditellurane], suppress PD-L1 expression on a variety of human and mouse malignant cells by inhibition of the α4β1 VLA-4 (very late antigen-4) integrin activity. As a consequence, pAkt and its downstream effector pNFκB are inhibited. VLA-4/pAkt/pNFκB signaling blockade was found crucial for decreasing PD-L1 expression. Moreover, the tellurium compounds had physiological effects. SAS augmented killing of human and mouse malignant cells by stimulated syngeneic splenocytes or CD8+ T cells. Furthermore, SAS prevented the development of chemoresistance in malignant cells. IL-10 production by tumor cells, the inhibition of which by AS101 we showed suppressed chemoresistance, is shown here to be closely associated with inhibition of VLA-4 and PD-L1 expression suggesting that AS101 and SAS compounds may, at least partially, induce chemosensitivity by virtue of inhibition of the VLA-4/IL-10/PD-L1 pathway. AS101 or SAS treatment of B16 melanoma-bearing mice decreased tumor cell PD-L1 expression, led to increased CD8+ T-cell infiltration into the tumors and tumor shrinkage. Combined treatment with αPD-1 antibody and either of the tellurium compounds significantly increased antitumor effects. These results collectively suggest that VLA-4 integrin signaling serves as a critical component of cancer evasion and is a potential target for cancer treatment. Furthermore, AS101 or SAS may be suitable for effective combinatorial cancer immunotherapy with αPD-1.
The Potential of Therapeutic Targeting of the TWEAK-Fn14 pathway in Atopic Dermatitis
Authors: Gupta Rinkesh Kumar , Fung Kai , Figueroa Daniela , Miller Jacqueline , Ay Ferhat , Croft Michael
Published: August 2023

Atopic dermatitis (AD) is a chronic skin inflammatory condition exhibiting a markedly thickened epidermis. Keratinocytes, the major cells of the epidermis, are a key target of inflammation and several cytokines including IL-13 and IL-22 have receptors expressed on these cells. Because Fn14 (TNFRSF12A), the receptor for the TNF family cytokine TWEAK (TNFSF12), is also expressed on keratinocytes, we examined the inflammatory potential of TWEAK alone, and together with IL-13 and IL-22, in controlling AD-signature gene expression in using RNA-seq. Transcriptomic analysis showed that TWEAK strongly upregulated many AD genes in human keratinocytes, which partially overlapped with those induced by IL-13 and IL-22. TWEAK also acted synergistically with IL-13 and IL-22 to exaggerate induction of AD-related genes. Correspondingly, cKO mice lacking Fn14 only in keratinocytes were protected from developing pronounced allergen-induced AD-like skin inflammation. Furthermore, mice treated therapeutically with anti-TWEAK exhibited a significantly reduced AD phenotype including less epidermal thickening combined with a strongly reduced Th2 response in the skin. Moreover, the therapeutic efficacy of TWEAK inhibition was similar to that resulting from blockade of IL-13. The data suggest that TWEAK shares pathogenic activity with IL-13 and IL-22 and can be considered as a potential therapeutic target in AD similar to IL-13.

Nil
Use of CD122+ natural killer cell precursors for superior anti-leukemia response
Authors: Krieg Carsten , Guglietta Silvia
Published: August 2023

Acute Myeloid Leukemia (AML) is an aggressive blood cancer in adults. Intensive induction therapy successfully induces a complete response in up to 80% of the adult patients but a fraction of them is refractory or relapses. The fraction of non-responsive or relapsing patients is especially high amongst medically unfit and older patients, who cannot undergo aggressive chemotherapy treatment. Therefore, offering patients failing on first line intensive chemotherapy and medically unfit and older patients an effective treatment option for long-term control or even cure of disease represents a significant yet unmet clinical need.

As a component of the innate immunity, NK cells have recently shown great promise for the treatment of patients with different malignancies. Here, we show that injection of human interleukin-2 (IL-2) complexes (IL-2cx) induce the de novo generation and massive expansion of NK cell precursors in vivo. Furthermore, IL-2cx-expanded NK cells exert effector functions with the capacity to control the growth of non-self MHC class I-deficient RMA-S lymphoma cells in vivo, while remaining tolerant towards MHC class-expressing self RMA cells.

In an experimental setup mimicking the clinical case of refractory patients after intensive AML induction therapy, IL-2cx treatment mediated strong anti-AML responses following haploidentical bone marrow transplantation without the need for adoptive transfer of donor-derived or in vitro autologous expanded NK cells. Thus, this study demonstrates that IL-2cx immunotherapy allows the in vivo generation and expansion of functionally mature NK cells to achieve long-term response in AML paving the way to an effective treatment option.

We are sponsored partly by the Cell Evaluation & Therapy Shared Resource, Hollings Cancer Center, Medical University of South Carolina (P30 CA138313), and an ACS-IRG to CK.
High Throughput Screening Identifies CDK Inhibitors as Inflammasome-Activating Vaccine Adjuvants
Authors: Weiss Adam M , Rosenberger Matthew G , Kim Jeremiah Y , Lopez Marcos A , Rowan Stuart J , Esser-Kahn Aaron P
Published: August 2023

Several FDA-approved adjuvant formulations, such as saponins and lipid nanoparticles, have been shown to mediate their adjuvant effects in part through the NLRP3 inflammasome. These formulations are among the most potent adjuvants in use, but they are limited by poor synthetic control and immunotoxic side effects. New small molecule adjuvants targeting the NLRP3 inflammasome would allow broader implementation of subunit- and nucleotide-based vaccines against poorly immunogenic antigens. In this work, we leverage high throughput screening of immunomodulatory compounds to identify cyclin dependent kinase (CDK) inhibitors as a novel class of inflammasome-activating small molecule adjuvants. CDK inhibitors are FDA-approved anti-cancer drugs which block cell cycle progression to prevent tumor proliferation. When co-administered with a Toll-like receptor (TLR) agonist, we observe that these inhibitors induce robust NLRP3- and Caspase-dependent IL-1β secretion in primary murine and human dendritic cells. When formulated with a TLR4 agonist and ovalbumin for use in vaccination, these compounds were found to enhance antibody production and antigen-specific CD8+ T cell proliferation relative to formulation controls. While greater work is needed to formulate and optimize these adjuvants, understanding the mechanism of CDK inhibitors’ adjuvanticity will both enhance our understanding of these compounds and expand their use case to vaccination.

Supported by grants from NIH (75N93019C00041, T32 GM008720) and DTRA (1-18-1-0052).
Characterization of a novel therapeutic antibody targeting peptide/HLA-E
Authors: Upchurch-Ange Katherine Christine , Oter Gizem , Gimlin Susanne , Weidanz Debra Wawro , Weidanz Jon
Published: August 2023

T-cell receptor mimic antibodies (TCRm) have become an attractive therapeutic modality group as they allow for the targeting of intracellular proteins presented by HLA, which may allow for a large potential of new targets. However, the polymorphic nature of classical HLA class I and findings of HLA downregulation in cancer cells might limit the use of these agents for cancer treatment. An alternative approach would be to target peptides presented by HLA-E, a highly monomorphic molecule often found upregulated in cancer cells. Thus, we aimed to generate a TCRm against HLA-E presenting the leader sequence peptide from HLA-G since both HLAs are often expressed in multiple cancer types. An antibody was identified from a naïve human phage library and further affinity matured using yeast display. The final clone, ABX-001, had an affinity constant (KD) of 7.9nM. Specificity studies using both peptide-loaded cells and tumor cell lines showed that ABX-001 only bound when both HLA-E and HLA-G were present. Extensive profiling was done on immune cells to also confirm that they did not bind ABX-001. Finally, ABX-001 was assessed for its ability to direct killing of target-positive tumor cell lines. We observed that cytotoxicity in CD8+ T cells was enhanced with the addition of an ABX-001-CD3 T cell engager. We also observed that ABX-001 can act as a checkpoint inhibitor by blocking the interaction of HLA-E with NKG2A in cytotoxicity experiments using NK cells. These data indicate that we can effectively target peptide/HLA-E and highlights its potential therapeutic applications in cancer treatment.
Functional DKK1 antibodies demonstrate antagonistic and tumor suppression activities
Authors: Wang Linya , Peterson Sean Michael , Lujan Ana , Chan Kara Y , Hu Cameron , Villalta Mouna , Yuan Tom Z , Han Zhen , Yang Marisa , Prabhu Vishwaas , Htoy Tammy , VanDyke Paul , Holliday Carson , Franco Hector , Wadhwa Hansika , Giang Hoa , Stafford Ryan , Axelrod Fumiko , Sato Aaron
Published: August 2023

Wnt signaling plays an important role in embryonic development and tumorigenesis. These biological effects are exerted by activation of the β-catenin/TCF transcription complex and consequent regulation of a set of downstream genes. DKK1 has been shown to be a potent inhibitor of Wnt signaling via competing Wnt binding to LRP5/6. DKK1 is tumorigenic in multiple cancer types and also immunosuppressive via MDSCs and NK cells. Emerging evidence indicates that DKK1 has been involved in T cell differentiation and induction of cancer evasion of immune surveillance by accumulating MDSCs. Consequently, DKK1 has become a promising target for cancer immunotherapy, and the mechanisms of DKK1 affecting cancers and immune cells have received great attention. With Twist’s precision DNA writing technologies, we have created phage display libraries with diversity greater than 1e10, and utilized machine learning model for optimal discovery. In this study, we discovered anti-DKK1 antibodies blocking the binding of DKK1 to the receptor. The in vitro functional assays showed that the interaction of Wnt to its receptor was restored in the presence of anti-DKK1 antibodies, resulting in TCF/LEF signaling upregulation. Moreover, anti-DKK1 antibodies induced immune cell activation, and led to tumor cell cytotoxicity. In addition, anti-DKK1 antibodies binding to different domains of DKK1 led to different functional effect. In vivo study indicated anti-DKK1 antibodies is potent in tumor regression. Our anti-DKK1 leads shows promising efficacy in cancer immunotherapy.
Canonical BAF Complex activity licenses CD8+ T cell effector fates
Authors: McDonald Bryan , Chick Brent , Ahmed Nasiha , Hargreaves Diana , Kaech Susan
Published: August 2023

CD8+ T cells provide a critical layer of host protection against all manner of pathogens and malignancies. Chromatin remodeling is a critical component of differentiation and acquisition of cytotoxic functions in activated CD8+ T cells, but the mechanisms by which remodeling is achieved in a site-specific way to promote appropriate effector cell fates are poorly understood. Here we identified a role for the ARID1A-containing canonical BAF (cBAF) complex in regulating chromatin accessibility and differentiation of antiviral effector CD8+ T cells. Arid1a was required for CD8+ T cell differentiation into terminal effector and tissue-resident memory cells, but was not required for the formation of long-lived circulating memory cells. Mechanistically, cBAF acted to promote chromatin accessibility at thousands of loci bound by important effector-associated TFs including BHLHE40, T-bet, and BATF. Understanding how chromatin remodeling is regulated in differentiating effector CD8+ T cells will yield insight into optimizing vaccines and adoptive cell therapies for cancer.

Supported by grants from NIH (F99 CA274688, R37 AI066232, R01 AI151123)
Inhibition of distinct glycolytic enzymes produces differential effects on CD4 T cell function
Authors: Godfrey Wesley H , Cho Kaho , Shanmukha Shruthi , Deng Jodie , Kornberg Michael
Published: August 2023

Introduction

When T cells are activated, they upregulate glycolysis and take on a requisite Warburg phenotype. In this study, we evaluated the effect of inhibiting two distinct glycolytic enzymes, GAPDH and PGAM, on CD4 T cell differentiation.

Methods/Results

Using the GAPDH inhibitor heptelidic acid, we found that GAPDH inhibition produces a potent anti-inflammatory phenotype in Th1 cells, significantly reducing IFNγ expression. We further showed that GAPDH inhibition produces the anti-inflammatory metabolite methylglyoxal, which is necessary for its anti-inflammatory effect. GAPDH inhibition also produced anti-inflammatory effects in vivo, as heptelidic acid significantly reduced disease severity and altered immune subsets in a therapeutic treatment paradigm in the MOG35–55 EAE model of multiple sclerosis. Furthermore, we showed that GAPDH inhibition enhances Treg polarization, while inhibiting PGAM, a downstream glycolytic enzyme, potently blocked Treg polarization. Additionally, we found that inhibition of PGAM increases serine biosynthesis and subsequently alters 1-carbon metabolism. We also found that PGAM gene expression is associated with Treg differentiation and response to immunotherapy in lung cancer patients.

Conclusion

Overall, our in vitro and in vivo results indicate that targeting the glycolytic enzyme GAPDH produces an anti-inflammatory phenotype via the methylglyoxal pathway, while targeting the downstream enzyme PGAM produces an opposite effect via regulation of serine synthesis. Our findings suggest that the functional consequences of glycolysis inhibition depend on the specific enzymes targeted, which represent promising novel therapeutic targets for immunological disease.

This work was supported by NIH/NINDS grant K08NS104266 and Conrad N. Hilton Foundation Marilyn Hilton Bridging Award for Physician Scientists grant 17316 to MDK. WHG was supported by NIH MSTP Grant T32 GM136577 and the American Association of Immunologists’ Careers in Immunology Fellowship Program.
Antioxidation protein Nrf2 modulates glutamine metabolism to regulate activation-driven expansion of CD4+T-cells
Authors: Tripathi Aprajita , Pant Anil , Bugbee Ashlyn , Choi Ben H , Pyaram Kalyani
Published: August 2023

Nutrient metabolism governs the effector functions like activation, expansion, and subset differentiation of CD4+T-cells, the orchestrators of adaptive immunity. The levels of reactive oxygen species (ROS), the metabolic byproducts of cellular metabolism are regulated by the antioxidation pathway, which is composed of Keap1 and Nrf2 proteins. We observed that, the expression of Nrf2 is temporally regulated post-activation in CD4+T-cells (high on day 1 and low on days 2 and 3) indicating its role in CD4+T-cell effector functions. It is yet unknown if and how the antioxidation pathway crosstalks with cellular metabolism to shape CD4+T-cell effector responses. Here, using mice with T-cell specific deletion of Keap1 (Keap1-KO) or Nrf2 (Nrf2-KO) we deciphered the effects of high and low levels of Nrf2 (antioxidation) levels, respectively in CD4+T-cells. Upon activation in vitro, Keap1-KO CD4+T-cells with constitutively high levels of Nrf2 showed higher proliferation, increased expression of early activation markers and elevated TCR-mediated signaling compared to wildtype (WT) cells. In contrast, decreased proliferation and lower TCR-mediated signaling was noted in activated Nrf2-KO CD4+T-cells. To dissect the underlying mechanisms, we performed metabolite analyses and activation in nutrient-limiting conditions. Interestingly, activated Keap1-KO CD4+T-cells show upregulated glutamine metabolism as measured by metabolic intermediates and rely on glutamine for optimum activation and increased expansion. Together, our findings identify a mechanism by which antioxidation protein Nrf2 metabolically reprograms CD4+T-cells and modulates their metabolic dependencies to support their activation and expansion.
Pyruvate Dehydrogenase Operates as an Intramolecular Nitroxyl Generator During Macrophage Metabolic Reprogramming
Authors: Palmieri Erika M , Holewinski Ronald , McGinity Christopher , Pierri Ciro , Maio Nunziata , Wink David , McVicar Daniel W
Published: August 2023

M1 macrophages “commit” to a glycolytic state when endogenous nitric oxide (NO) reprograms mitochondrial metabolism by limiting aconitase (Aco)-2 and pyruvate dehydrogenase (PDH). Here we provide evidence that NOdirectly targets the PDH complex using lipoate to generate nitroxyl (HNO). PDH E2-associated lipoate is modified in NO-rich macrophages while the PDH E3 subunit is irreversibly inhibited. Mechanistically, we show that lipoate facilitates NO-mediated HNO production that interacts with thiols forming modifications resistant to reduction. Consequently, we reveal a macrophage signature of proteins with reduction resistant modifications, including E3, and identify a subset of potential HNO targets. Consistently, recombinant E3 is modified in HNO-dependent manner at Cys477 and Cys484, and molecular modeling shows these cause impairment of functional E3 homodimer formation. Our work demonstrates that HNO is produced physiologically, and its presence is peculiar to the lipoate rich PDH complex facilitating irreversible modifications critical in NO-dependent metabolic rewiring.
Androgen Receptor regulates Ptpn22
Authors: Lee Jean A , Yurkovetskiy Leonid A , Frommer Laura , Strong Zoe , Chang Anthony , Khan Aly A , Kahaly George J , Chervonsky Alexander V
Published: August 2023

The need to rear the offspring necessitated the evolution of female’s immune system to mount stronger responses than males. One consequence of it is that major autoimmune diseases manifest in a sex biased manner with a predominance in females. However, mechanisms underlying the bias have not been clearly defined. We investigated the molecular mechanisms by which male sex hormones (androgens) regulate αβ T cell function in sex biased autoimmunity. We identified Ptpn22, a gene encoding a lymphoid-specific phosphatase that negatively regulates proximal TCR signaling events, as a direct target of a transcription factor Androgen Receptor (AR) in T cells. In T cells isolated from sham-operated or castrated mice, transcription of Ptpn22 correlated with the presence of androgens. Furthermore, stimulation of Jurkat T cells with AR agonist resulted in induction of Ptpn22 transcription only in the presence of AR. Deletion of Ptpn22 in the B6.NZM mouse model of Systemic Lupus Erythematosus (SLE) led to exacerbation of autoimmunity accompanied by the loss of sexual dimorphism. We identified an androgen response element (ARE) in the 5′ UTR of Ptpn22 and found that its CRISPR/Cas9-mediated mutation enhanced both in vitro and in vivo the autoreactivity of T cells in NOD/ShiLtJ mouse model of Type I Diabetes (T1D). In humans, PTPN22 R620W mutation associated with several autoimmune disorders inversely correlated with the sexual dimorphism of autoimmunity. Thus, the ‘wild-type’ common allele of PTPN22 is likely to be involved in sex bias in autoimmune conditions.

NIH grant R01 AI 127411
Early peripheral B cells in inactive SLE are transcriptionally and epigenetically primed for T-dependent activation
Authors: Rose James R , Ishii Yusho , Hicks Sakeenah , Scharer Christopher D , Sanz Ignacio , Boss Jeremy
Published: August 2023

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease characterized by repeated episodes of increased disease activity followed by periods of inactivity and remission. Increased titer of anti-dsDNA autoantibodies is often associated with active disease and the proliferation of autoreactive B cells are thought to play a major role in the progression of disease. In this study we investigated the transcriptional and epigenetic profile of early peripheral B cells—transitional (T1T2) and resting naïve (rN)—in inactive and active disease from a large cohort (n=46) of African American SLE patients. Our results show that the transcriptome of these B cells show a dysregulated signature as previously reported, but this signature is distinct between inactive and active SLE. Active disease is characterized by a greater number of differentially expressed genes (DEG) compared to healthy controls (HC), however, rN B cells in inactive disease patients express high levels of genes associated with T-dependent activation, IL4 signaling, and germinal center reactions. Chromatin accessibility across the genome of inactive disease B cells show that they share a subset of differentially accessible regions (DAR) from HC that are similarly dysregulated in both inactive and active flaring individuals, but inactive SLE B cells also have acquired several hundred unique DAR regions—some of which correspond with motifs or loci encoding important B cell development transcription factors. These findings suggest a distinct profile in early B cells from inactive disease patients that remains present during disease remission and may predispose these cells towards T-dependent activation as a response to long-term disease.

Supported by grants from NIH (T32 GM0008490, 3U19 AI110483-08S2 8744)
IL2 signaling and Foxp1 expression maintain Treg identity in the absence of Foxp3
Authors: Hill Charlotte Nicole , Zhong Yuelin , Zemmour David , Charbonnier Louis-Marie
Published: August 2023

Regulatory T cells (Treg) have a pivotal role in tolerance and immune homeostasis. In autoimmunity, Treg are destabilized and degenerate, loosing Foxp3 expression and compromising their regulatory function, while gaining a T effector cell (Teff)-like phenotype. Upon Foxp3 loss-of-function, Treg development gives rise to a heterogeneous population of Treg cells (ΔTreg). ΔTreg retain the core Treg transcriptome and epigenetic imprint and can be classified based on the expression of the high-affinity IL-2 receptor-alpha, CD25, and Foxp3 locus activity. We herein propose a stepwise degeneration driven by a decrease in IL2/CD25 signaling and subsequent loss of Foxp1 expression.

CD25+ ΔTreg expression profile, as compared to the one of CD25− ΔTreg cells, presented increased levels of genes typically expressed in Treg, such as Il2ra and Il10. Consistently, CD25− ΔTreg were biased toward Th1, presented impaired suppressive capacity, and downregulated Foxp1. Treatment with IL2/aIL2 and STAT5b gain of function upregulated CD25, decreased Th1 bias and inflammation, supporting the role of IL2 signaling halting the passage from CD25+ to CD25− ΔTreg. ΔTreg-specific depletion of Foxp1 led to a steep decrease in the frequency of ΔTreg, increased T-bet expression, and a reduction in the Foxp3 locus activity, suggesting that Foxp1 loss drives ΔCD25− ΔTregs into a terminally degenerated state. Our results indicate that IL2 signaling is sufficient to maintain ΔTreg regulatory capacity, while Foxp1 expression is necessary to stabilize their identity and prevent terminal degeneration. We are currently doing scRNAseq and ChIPseq to elucidate the molecular mechanisms by which IL2 signaling and Foxp1 maintain Treg identity in the absence of Foxp3.

National Institutes of Health grant R01AI153174
PDGFRα promotes organ resistance to immune-mediated damage
Authors: Rosetti Florencia , Meléndez-Pérez Diana Laura , de la Cruz Abigail , Larios-Orozco Emiliano , González-Yáñez Marysol , Furuzawa-Carballeda Janette , Villanueva Raul , Cevallos Cristina , Madera-Salcedo Iris Karina , Crispin Jose C
Published: August 2023

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that exhibits great clinical heterogeneity. Development and clinical expression of autoimmune diseases is strongly influenced by genetic variants. The single nucleotide polymorphism (SNP) rs1364989, located close to PDGFRA, has been associated with the development of lupus nephritis through an unknown mechanism. Because PDGF, through its α and β receptors, regulates tissue response to inflammation, we hypothesized that rs1364989 could modulate the behavior of renal cells in the context of immune-mediated glomerulonephritis.

To determine whether rs1364989 affects the expression of PDGFRα, we analyzed umbilical cord mesenchymal cells from carriers of risk and protective alleles. We observed that the presence of the risk allele was associated with reduced PDGFRα expression. Next, we analyzed whether inflammatory mediators could modulate PDGFRα expression in in vitro and in vivo settings. We documented that its levels increase during acute inflammation in a murine model of glomerulonephritis. In vitro, this effect could be reproduced by IL-17. Absence of PDGFRα increased kidney damage during immune complex-induced glomerulonephritis. An analogous effect was observed in joints of mice with collagen-induced arthritis.

Our results demonstrate that the SLE-associated allele of rs1364989 reduces the expression of PDGFRα and that this receptor may activate a pathway that protects tissues in response to local inflammation. These findings highlight the importance of tissue resilience as a factor that can protect an organ from inflammatory damage in the setting of autoimmunity.

This work was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT), Mexico
Lymphoid Follicle Transcriptome Reveals Maladaptive Immune Responses In Severe Chronic Obstructive Pulmonary Disease (COPD)
Authors: Rojas-Quintero Joselyn J , McKenna Neil J , Ochsner Scott A , Cala-Garcua Juan D , Kheradmand Farrah , Rosas Ivan O , Polverino Francesca
Published: August 2023

Rationale

Increases in the number and size of lymphoid follicles (LFs) are present in severe stages of COPD and have not been fully characterized. HLA complex is crucial in enabling the immune system to recognize “self” versus “non-self” antigens and plays a major role in immune responses to cigarette smoke exposure.

Methods

Lung follicles from COPD patients with milder (GOLD I–II) and more severe (GOLD III–IV) COPD were sampled using GeoMX® Digital Spatial Transcriptional Profiling. Data were analyzed and normalized using GeoMx® DSP Analysis Suite.

Results

Pathway analysis showed that B cell activation pathways had the highest fold enrichment. GOLD I–II compared to GOLD III–IV COPD patients had the highest expression of HLA-C and its ligands (KIR2DS2, KIR2DS1, KIR3DL1; p<0.0001) which facilitate CD8-mediated cytotoxicity, and HLA-G and its receptor LILRB4 (p=0.01) which are key modulators of immune tolerance. GOLD III–IV COPD patients had the highest HLA enrichment in LFs, with upregulation of transcription factors (HIF1A, IRF1, NFYBand NFYC) and assembly components (CALR, TAPBP, ERP27, MARCHF1, B2M, and CIITA) of HLA (p<0.01). Compared to GOLD I–II, LFs from GOLD III–IV COPD patients had much higher expression of HLAs involved in autoimmune responses to self-antigens and adaptive immune evasion, such HLA-DRA, HLA-DRB1, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-E and HLA-B (p<0.0001).

Conclusions

We show for the first time that LFs from patients with severe COPD are characterized by expression of HLAs associated with immune evasion and autoimmune responses, whereas LFs in milder stages of COPD are characterized by induction of immune responses controlled by tolerance mechanisms

R01 HL 149744-04/06
Microbiota dampen type 2 immunity by epigenetically restricting tuft cell differentiation
Authors: Eshleman Emily M , Rice Taylor , Potter Crystal , Woo Vivienne , Field Sydney , Hashimoto-Hill Seika , Waddell Amanda , Engelman Laura , Finkelman Fred D , Alenghat Theresa
Published: August 2023

Type 2 immune responses at mucosal surfaces are both required to expel helminthic parasites and central to allergic disease pathogenesis. Tuft cells are central regulators of type 2 immunity that instruct downstream type 2 innate lymphoid cells (ILC2s). Although the microbiota are known to shape immune responses, the impact of commensal microbes on regulation of tuft cell-dependent immunity remains poorly understood. Here, we find that butyrate-producing commensal bacteria suppress tuft cell-dependent type 2 immunity in the intestine. Butyrate suppression of tuft cells required the microbiota-sensitive epigenetic modifying enzyme histone deacetylase 3 (HDAC3), suggesting that HDAC3 may promote tuft cell-dependent immune defense. Consistent with this, epithelial-intrinsic HDAC3 actively stimulated tuft cell responses, was required to initiate the tuft cell-ILC2 feed-forward response to Nippostrongylus brasiliensis infection, and promoted expulsion of this helminthic parasite. Furthermore, butyrate epigenetically restricted stem cell differentiation into tuft cells, and inhibition of HDAC3 in adult mice and human intestinal organoids was sufficient to block tuft cell differentiation. Collectively, these data reveal a microbiota-sensitive epigenetic pathway in stem cells that dampens tuft cell mediated type 2 immunity, and highlight a new level of regulation through which commensal bacteria can calibrate mucosal inflammation.

This research is supported by the National Institutes of Health (DK114123, DK116868 to T.A., and F32AI147591 to E.M.E.), and a Kenneth Rainin Foundation award to T.A. T.A. holds an Investigator in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund. This project is supported in part by PHS grant P30 DK078392 and the CCHMC Trustee Award and Procter Scholar’s Program.
MHCII Expression by Intestinal Goblet Cells in Immune Homeostasis
Authors: Barrios Bibiana E , John Vini , Floyd Alexandria , Davis Jazmyne , Udayan Sreeram , Schill Ellen , McDonald Keely , Newberry Rodney D
Published: August 2023

As the primary barrier in the gut, intestinal epithelial cells (IECs) are positioned to promote tolerance and immunity against pathogens. MHCII is highly expressed on IECs, however MHCII expression on epithelial cell subsets is largely unexplored. Goblet cells (GCs) are specialized epithelial cells that have critical role in barrier maintenance and immunity. GC can form Goblet cells associated passage (GAPs) and deliver luminal antigens to lamina propria (LP) antigen presenting cells (APC) to induce adaptive immune responses. Here, we report that MHCII is highly expressed on small intestinal GCs as compared with other IECs and colon IECs, which do not form GAPs in the steady state. Moreover, expression of coinhibitory and costimulatory molecules by GCs was higher when compared with other IECs. Interestingly, flow analysis and quantification of MHCII on CD45.2+ MHC-II−/− donor-derived leukocytes showed MHCII acquisition by donor-derived small intestinal lamina propria (siLP) APCs 7 weeks after BM-transplant into CD45.1+ recipients. Indeed, carbachol, which induces GAPs formation resulted in increased MHCII acquisition by CD45.2+MHC-II−/− donor-derived siLP MNPs. Furthermore, selective deletion of MHCII on GCs, resulted in a significant reduction of transferred dietary antigen specific OT-II+CD45.1+CD4+ T cells in mLN in a model of oral tolerance. Bulk RNAseq analysis on the small intestine of mice with GC deletion of MHCII revealed an expansion of B cell responses and significant increase in IgA+ cells in Peyer’s patches. This data suggests that GC expression of MHCII has a role in antigen-specific responses to luminal antigens and in regulating intestinal homeostasis.

Supported by grants from NIH
T cell receptor signaling mediates enhanced IFNγ production by γδ intraepithelial lymphocytes in response to type I interferon
Authors: Fischer Matthew , Jia Luo , Edelblum Karen
Published: August 2023

Intraepithelial lymphocytes (IEL) expressing the γδ T cell receptor (TCR) provide a rapid response to limit enteric pathogen invasion. Despite constant γδTCR triggering in vivo, these IELs remain immunologically quiescent until their activation threshold is surpassed. Activated γδ IELs limit viral replication by producing type I interferons (IFN), such as IFNα, yet the extent to which IFNα activates γδ IELs remains unknown. To this end, murine small intestinal γδ IELs were stimulated ex vivo with 1 ug/mL αCD3, 10 ng/mL IFNα or both for 5 h. We observed a 26%±10 increase in IFNγ+ γδ IELs treated with αCD3 and IFNα compared to αCD3 treatment alone (p<0.0001) and a 2-fold increase in IFNγ MFI (p<0.0001). Using Nur77-GFP mice, we found that only Nur77+ γδ IELs produced IFNγ (p<0.0001), indicating that TCR signaling is necessary for IFNα-induced effector function. Suboptimal αCD3 stimulation (0.1 ug/mL) was sufficient to increase the frequency of IFNγ+ γδ IELs following IFNα exposure relative to αCD3 alone (p<0.001). To interrogate the molecular mechanism involved in IFNα co-stimulation, γδ IELs were treated with PMA and/or ionomycin in the presence or absence of IFNα. Ionomycin and IFNα increased the frequency of IFNγ+ γδ IELs by 16%±7 compared to ionomycin alone (p<0.0001), whereas PMA had no effect. Pharmacological inhibition of NFAT signaling (10 uM INCA6) abrogated both TCR- or IFNα-induced γδ IEL IFNγ production (p<0.01 or p<0.0001, respectively). Lastly, we observed that the co-stimulatory effect of IFNα was lost in STAT4-deficient γδ IELs compared to WT (p<0.0001). Together, these data indicate that low level TCR signaling through NFAT is required for IFNα to rapidly enhance γδ IEL IFNγ production in a STAT4-dependent manner.

This work was supported by the National Institutes of Health R01 DK119349, the New Jersey Commission on Cancer Research, Busch Biomedical Research grant and the RBHS Chancellor Scholar Award (KLE).
CaV3.2 links mast cells, tryptase and pain
Authors: Fairlie Samantha E , Chovanec Jack , Liu Yihui , Walitt Brian , Saligan Leorey , Lyons Jonathan J
Published: August 2023

Hereditary a-tryptasemia (HaT) is a genetic trait defined by elevated serum tryptase caused by increased TPSAB1 copy number and associated with multiple phenotypes including pain. CACNA1H encoding the voltage-gated calcium channel CaV3.2 overlaps the tryptase locus where a partial gain-of-function (GOF) haplotype containing 3 linked variants has been shown to be co-inherited with HaT in ~2/3 of individuals. While CaV3.2 is expressed in neurons where it contributes to pain perception, expression has also been observed in other cells. Mast cells are the principal source of tryptase, exist in proximity with nerves, and have been implicated in pain sensation. We set out to investigate links between these variants, mast cells, and pain. Performing tryptase and CACNA1H genotyping via ddPCR in a cohort of fibromyalgia patients we found that HaT was enriched compared to the general population (p=0.029). However, the CACNA1H haplotype was universally present, suggesting these variants were driving the association. In support of this observation, enrichment of the GOF variants was also observed among those without HaT. Using flow cytometry, immunoblotting, ddPCR, and immunofluorescence, we found LAD2 cells express CaV3.2 where it co-localizes at the cell surface with calnexin and confirmed expression in primary cultured human mast cells. To our knowledge, this is the first report of a voltage-gated calcium channel on mast cells. While calcium flux is critical for mast cell activation, mechanisms driving voltage-gated channel activation remain to be elucidated. Future studies will help unravel the mechanisms linking mast cell activation and pain pathways enabling the development of therapies for these commonly linked clinical phenotypes.

Supported by grant from DIR NIAID NIH, grant: ZIA AI001192
BLOC1S1: An Unexpected Regulator of Th2 Cell-driven Inflammatory Responses
Authors: Sharma Rahul , Wu Kaiyuan , Han Kim K , Singh Komudi , Sack Michael N
Published: August 2023

BLOC1S1 is an adaptor protein that modulates endosome-lysosome function as a component of numerous multiprotein complexes. We postulated that its modulation in CD4+ T cells may help us uncover the role of endosome-lysosome biology in adaptive immunity. In primary CD4+ T cells BLOC1S1 cre-recombinase knockout resulted in the preferential induction of Th2 compared to Th1 and Th17 cytokine production following T cell receptor activation. As atopic dermatitis is an allergic inflammatory skin disease characterized by the production of the type 2 cytokines in the skin we evaluated the effect of CD4+ T cell BLOC1S1 KO vs controls in calcipotriol-induced atopic dermatitis in mice. The absence of BLOC1S1 exhibited exacerbated atopic dermatitis accompanied with significantly elevated plasma IgE, skin eosinophilia, elevated type 2 responses, and inflammatory pathology relative to wild-type mice. The transcription factor Gata-3 is expressed in Th2 but not Th1 cells and plays a critical role in Th2 differentiation and atopic dermatitis. BLOC1S1 deficient CD4+ T cells showed enhanced nuclear factor kB (NF-kB), Gata3 and LC3 positive autophagosomes. Inhibition of NF-kB activity prevented Gata-3 expression, LC3 positive autophagosomes and Th2 cytokine production in BLOC1S1 −/− CD4+ T cells. This mouse model will now give us a platform to explore the role of endo-lysosomal biology in Th2 polarization and activation.

NHLBI Division of Intramural Research (MNS-ZIA-HL005102)
Therapeutic efficacy and mechanism of a solubilized Sturgeon oil in a mouse model of house dust mite-induced atopic dermatitis
Authors: Lee Hee-Sung , Park Ji-Hyun , Park Chan-Su , Lee Chong-Kil
Published: August 2023

The therapeutic effect and action mechanism of solubilized Sturgeon oil (SSO) against atopic dermatitis (AD) were examined in NC/Nga mice. SSO was prepared from the fish oil obtained from Sturgeons using physical solubilization processes. AD was induced by topical application of house dust mite (HDM) extract on the dorsal skin of NC/Nga mice, and then treated with SSO. SSO exerted potent therapeutic activity on AD symptoms. Histological analysis revealed that SSO reduced epidermal hyperplasia and suppressed infiltration of mast cells and eosinophils in the dorsal skin. In addition, SSO significantly reduced the serum total IgE and HDM-specific IgE levels in AD-induced NC/Nga mice. Analysis of the dorsal skin tissues showed that SSO significantly downregulated the expression levels of IL-4, 5, 13, 17A and COX-2, while upregulated the expression levels of IL-10 and filaggrin. In vitro studies also showed that SSO suppressed the production of Th2 cytokines including IL-4, 5 and 13, while increasing the production of IL-10, from anti-CD3/CD28-stimulated Th2 cells. These results show that SSO exerts therapeutic effect on AD in NC/Nga mice through Th2-related immunomodulatory activities.
A mechanism for the elimination of IgE plasma cells
Authors: Wade-Vallance Adam Kevin , Yang Zhiyong , Libang Jeremy B , Robinson Marcus J , Tarlinton David M , Allen Christopher D C
Published: August 2023

The proper regulation of IgE production safeguards against allergic disease, highlighting the importance of mechanisms that restrict IgE plasma cell (PC) survival. IgE PCs have unusually high surface B cell receptor (BCR) expression, yet the functional consequences of ligating this receptor are unknown. Here, we found that BCR ligation induced BCR signaling in IgE PCs followed by their elimination. In cell culture, exposure of IgE PCs to cognate antigen or anti-BCR antibodies induced apoptosis. IgE PC depletion correlated with the affinity, avidity, amount, and duration of antigen exposure and required the BCR signalosome components Syk, BLNK, and PLCγ2. In mice with a PC-specific impairment of BCR signaling, the abundance of IgE PCs was selectively increased. Conversely, BCR ligation by injection of cognate antigen or anti-IgE depleted IgE PCs. These findings establish a mechanism for the elimination of IgE PCs through BCR ligation. This has important implications for allergen tolerance and immunotherapy as well as anti-IgE monoclonal antibody treatments.

This research was supported by the National Institute of Allergy and Infections Diseases of the National Institutes of Health under Award Number R01AI130470; the Program for Breakthrough Biomedical Research, which is partially funded by the Sandler Foundation; the Sandler Asthma Basic Research Center; and the Cardiovascular Research Institute at the University of California, San Francisco. C.D.C.A. was a Pew Scholar in the Biomedical Sciences, supported by The Pew Charitable Trusts. A.K.W-V was supported by a Doctoral Foreign Study Award from the Canadian Institute of Health Research, funding reference number DFD-170769. D.M.T. and M.J.R. were funded by National Health and Medical Research Council (NHMRC) Australia by an Investigator Award (APP1175411) and Ideas Grant (APP1185294), respectively. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.
Histamine 3 receptor antagonism mitigates neuroinflammation and depression-like behavior in a mouse model of food allergy
Authors: La Mae Germundson-Hermanson Danielle , Nagamoto-Combs Kumi
Published: August 2023

Histamine (HA) is a major allergic response mediator as well as a neuromodulatory neurotransmitter and a blood-brain barrier (BBB) regulator. Consequently, dysregulation of the central histaminergic system could offset brain homeostasis and function. Indeed, mood and behavioral disorders are often comorbid with conditions that elevate systemic HA, such as mastocytosis and allergy. We have previously shown elevated levels of brain HA and its receptor subtype H3 (H3R) in a mouse model of cow’s milk allergy (CMA). Additionally, neuroinflammation and cortical demyelination were observed in association with depression-like behavior in CMA mice. However, whether the elevated HA and H3R were responsible for the development of these cellular and behavioral pathologies is unknown. We hypothesized that the elevated brain HA leads to central histaminergic dysfunction through H3R, ultimately resulting in cortical demyelination and aberrant behaviors. To investigate the role of H3R, CMA mice were treated daily with thioperamide, a selective BBB-permeable H3R antagonist, to block the action of HA through this receptor. Despite the production of allergen-specific IgE, thioperamide prevented depression-like behavior. Thioperamide also ameliorated CMA-associated cortical demyelination and axonal loss compared to saline-treated mice. Our findings strongly suggested that H3R signaling contributed to cortical demyelination and the development of depression-like behaviors in our CMA mice. Targeting HA production or signaling may provide a therapeutic strategy to reduce the risk of neurodegeneration and associated brain dysfunction in susceptible individuals.

Supported by grants from NIH (1R01AI168563-01, P20GM113123-06, and P20GM103442).
Identification of BST2 as a biomarker for alopecia areata in both mice and humans
Authors: Velasquez Cristina , Coast Jake
Published: August 2023

Alopecia areata is an autoimmune disease characterized by aberrant non-scarring hair loss. 147 million people worldwide are currently diagnosed with alopecia areata with a lifetime incidence of 2.1%. The hair follicle is a site of immune privilege however in alopecia areata, CD4+ helper T cells and CD8+ cytotoxic T cells infiltrate and attack the hair follicle. To better understand the initiating mechanisms surrounding alopecia areata, we examined the function of epidermal γδ T cells and dermal macrophages in the initiation of or protection from the disease. We show that epidermal γδ T cells and keratinocytes are elevated in the skin of C3H/HeJ mice with alopecia areata. Analysis of publicly available single cell RNA sequencing data delineated the top differentially expressed genes of epidermal γδ T cells and dermal macrophages. Bone marrow stromal antigen 2 (BST2) is significantly upregulated among both cell types and is a novel gene associated with alopecia areata. Using immunofluorescence microscopy we determined that a small population of hair follicle-associated epidermal γδ T cells express BST2 in C3H/HeJ mice and C57Bl/6J mice without alopecia areata. We have also identified a significant increase in BST2 expression in skin sections from mice affected by alopecia areata. BST2+CD11b+ macrophages upregulate genes associated with antiviral activity indicating an association between the pathogenesis of alopecia areata and infectious viral particles. BST2+ epidermal γδ T cells upregulate genes associated with FAK signaling in alopecia areata, which indicates a role for FAK in the increased number of epidermal T cells. These studies identify a role for BST2 in alopecia areata that is mediated by epidermal γδ T cells and macrophages.
Anti-PD-1 antibodies recognizing the membrane-proximal region are agonists that can stimulate immunosuppressive activity of PD-1
Authors: Ohta Akio , Suzuki Kensuke , Tajima Masaki , Tokumaru Yosuke , Oshiro Yuya , Nagata Satoshi , Kamada Haruhiko , Kihara Miho , Nakano Kohei , Honjo Tasuku
Published: August 2023

The success of immunoenhancing anti-PD-1 blocking antibodies in cancer treatment indicates the biological significance of PD-1-dependent immunoregulation in the human immune system. Active stimulation of PD-1 is potentially an effective approach in treating various inflammatory disorders; however, agonistic anti-PD-1 antibodies stimulating the immunosuppressive activity are not clearly defined. Here, by assessing the biological activities of anti-human PD-1 mAbs, we have identified functional anti-PD-1 agonists in the antibody group recognizing the membrane-proximal extracellular region (MPER). MPER recognition was characteristic to anti-PD-1 agonist mAbs and distinct from blocking mAbs binding to the membrane-distal region of PD-1. The agonistic activity was mediated by Fc receptor-dependent co-ligation of PD-1 molecules. In vivo anti-inflammatory efficacy of the PD-1 agonist in murine disease models suggests its clinical potential to various inflammatory disorders including autoimmune diseases.

Meiji Seika Pharma, Co., Ltd. Foundation for Biomedical Research and Innovation at Kobe