bluuebunny/crossref_metadata_2025 huggingface dataset:
Generation of autoreactive CD8 T cell in a mouse model of alopecia areata
Authors: Lee You Jeong , Lee Hee Eun
Published: August 2023

Alopecia areata (AA) is the most frequent form of an organ-specific autoimmune disease in humans mediated by NKG2D+ CD8 T cells destroying hair follicles. C3H/HeJ mice naturally develop AA similar to humans, and we found it accompanies the expansion of exhausted phenotype (CD101+ Tim3+) memory CD8 T cells not only in the skin and skin-draining lymph nodes (SDLNs), but also in the liver and lungs. Before the disease induction, we found these CD8 T cells are activated in the lungs and liver, suggesting they are pre-activated in these organs. In the skin, expanded CD8 T cells additionally upregulated CD39, PD-1, and CTLA-4, which produced less IFNg than those in the SDLN, liver and lungs when stimulated. These features suggest that autoreactive CD8 T cells in the skin are exhausted by chronic antigenic stimulation. However, they did not upregulate TOX, a hallmark of T cell exhaustion. Intriguingly, CD44high NKG2D-expressing CD8 T cells appeared in the thymi of aged C3H/HeJ mice before disease induction. These cells express TBET and EOMES without overt expansion of PLZF-expressing T cells. Collectively these features suggest AA is mediated by autoreactive activation CD8 T cells that are not explained by previously known mechanisms.

This research was supported by the National Research Foundation of Korea (NRF-2022R1A2C1007692)
The role of ST8Sia6 in braking inflammation in Inflammatory Bowel Disease
Authors: Crotts Sydney B , Antonetti Regina , Shapiro Michael , Rajcula Matthew , Hammer Brady , Mai Elissa , Shapiro Virginia
Published: August 2023

Inflammatory bowel disease (IBD) including ulcerative colitis (UC) and Crohn’s disease (CD) affects between 1–3 million people in the United States and costs an estimated $11–28 billion in national healthcare costs. Patients present with symptoms including diarrhea, rectal bleeding, and fatigue. Characterization of molecules that regulate IBD is critical in understanding pathogenesis. ST8Sia6 is a sialic acid transferase that adds terminal a2,8 disialic acids to cell surface glycoproteins, and we have found that ST8Sia6 knockout (KO) mice exhibit increased spontaneous small bowel (SB) lamina propria CD4 TH1 polarization, increased inflammatory cytokine production, decreased IgA class switching of SB plasma cells, and alterations of the SB innate lymphoid cells, indicative of gut inflammation. Moreover, adult ST8Sia6 KO mice have shortened small and large bowels, likely due to baseline inflammation. These mice do not exhibit overt baseline symptoms, but ST8Sia6 KO mice exhibit increased weight loss and decreased survival when challenged with dextran sodium sulfate (DSS)-induced colitis, which was also enhanced when co-housed with wildtype (WT) mice. DSS-challenged ST8Sia6 KO mice exhibit increased histological damage and immune infiltration of the gut as compared to WT controls. Accelerated DSS-induced colitis is also observed when ST8Sia6-deficient bone marrow is transplanted into WT recipients, indicating that the effect is mediated specifically by immune cells. This data demonstrates a role for ST8Sia6 in regulating the host immune response to IBD, and further elucidating this role will aid in developing a mechanistic understanding of IBD pathogenesis and in understanding potential immunotherapies for the disease.

R01CA243545, 5T32DK124190-03
Inducing/expanding Tregs in vivo to suppress autoimmunity using a new bispecific therapeutic containing IL-2 and TGFβ
Authors: Jackson Nicholas M , Hoft Stella , Ford Eric , Yakobian Natalie , Girgis Natasha , DiPaolo Richard
Published: August 2023

The objective of this study was to test a new injectable therapeutic, CUE-401, that is designed to induce Foxp3 expressing Tregs by signaling through both IL-2 and TGFβ receptors. Unlike IL-2 therapies that activate and expand existing Tregs, this biotherapeutic was designed to both induce new subsets of iTregs from naïve autoreactive CD4+ T cells and to activate and expand existing Tregs. Experiments were performed to determine the biological activities of CUE-401 both in vitro and in vivo after administering to mice with active and ongoing autoimmunity. The in vitro results showed that stimulating naïve CD4+ T cells in the presence of CUE-401 was effective at inducing Foxp3 expressing iTregs that were phenotypically and functionally indistinguishable from iTreg induced with recombinant IL-2 and TGFβ. When injected into mice, CUE-401 was effective at increasing Foxp3+Tregs. Unlike mice treated with IL-2 complexes, CUE-401 did not induce proliferation of autoreactive T cells. Single cell RNA sequencing revealed that CUE-401 induced a subset of Tregs that shared many transcripts known to be expressed by TGFβ-induced iTregs. Finally, CUE-401 induced iTregs were effective at suppressing organ specific autoimmunity. These studies identify a new injectable therapeutic, and new approach to induce and expand Tregs in vivo to suppress autoimmunity and other inflammatory diseases.
Investigating the interactions between regulatory T cells and cancer-associated fibroblasts and their role in lung cancer progression
Authors: Ringham Olivia Rose , Loffredo Lucas F , Arpaia Nicholas
Published: August 2023

The tumor microenvironment (TME) is a highly complex multicellular system and has been shown to play a fundamental role in dictating tumorigenesis. High expression of regulatory surface molecules, cytokines, and chemokines in fibroblast niches within lung tumor stroma, paired with their colocalization with tumor infiltrating lymphocytes (TILs), suggests that cancer-associated fibroblast (CAF) niches may alter the functional response of TILs and influence the anti-tumor immune response. Here, utilizing a transcriptomic approach, we have uncovered a distinct CAF population, which we term “iCAFs,” that is enriched for immunomodulatory gene signatures, T cell ligands, and chemoattractants. This population expresses a unique set of cell surface molecules that permit their identification and isolation from lung tumor tissue. Given the demonstrated importance of Tregs in suppressing anti-tumor immunity, we chose to evaluate how Tregs may be interacting with iCAFs within the TME of lung adenocarcinoma. We uncovered significant colocalization patterns of Foxp3+ Tregs with iCAF—a pattern which was found to be unique for Tregs when compared to conventional CD4+Foxp3− T cells. Moreover, the Treg to conventional CD4+ T cell ratio is significantly decreased in tumor tissue relative to normal adjacent tissue and healthy controls. Our analyses demonstrate that Tregs preferentially associate with iCAF populations within the tumor, leading us to hypothesize that interactions between Tregs and iCAFs may, in turn, influence Treg trafficking, phenotype and function within the TME.

T32AI106711
Sensory Nerves inhibit the development of protective anti-melanoma immune response and formation of tertiary lymphoid structures
Authors: Vats Kavita , Kruglov Oleg , Sahoo Bikram , Zhang Jiying , Sonam Vishal , Chandran Uma , Shurin Galina V , Skum Pavel , Shurin Michael R , Strokus Walter J , Bunimovich Yuri M L
Published: August 2023

Peripheral nervous system (PNS) is an important component of the tumor microenvironment (TME). It has been established that autonomic innervation plays an important role in cancer. However, the effect of the afferent (sensory) neurons on tumor progression remains unclear. Utilizing surgical and chemical skin sensory denervation methods, we showed that afferent neurons supported the growth of melanoma tumors in vivo and demonstrated that sensory innervation limited the activation of effective antitumor immune responses. Precisely, sensory ablation enhanced leukocyte recruitment into tumors, with decreased presence of lymphoid and myeloid immunosuppressive cells and increased activation of T-effector cells within the TME. Denervation further increased T-cell clonality and expanded the B-cell repertoire in the TME. Furthermore, Depletion of CD8+ lymphocytes prevented denervation-restricted tumor growth. Importantly, denervated tumors contained higher densities of mature high endothelial venules (HEV) associated with the formation of classic tertiary lymphoid structures (TLS), which associate with a more favorable melanoma prognosis. Transcriptional profiling of human melanomas confirmed our findings in murine melanoma models. Specifically, we observed that gene signatures of inflammation and the content of neuron-associated transcripts inversely correlated in human primary cutaneous melanomas, with the latter representing a negative prognostic marker of patient overall survival.. Our results suggest that tumor-associated sensory neurons negatively regulate the development of protective antitumor immune responses within the TME, thereby providing a new therapeutic target for melanoma treatment.
PSGL-1 deficiency eradicates orthotopic pancreatic ductal adenocarcinoma tumors in combination with anti-PD-1 therapy and enhances T cell anti-tumor immunity
Authors: Roy Sreeja , Hope Jennifer L , Zhang Yijuan , Palete Ashley B , Faso Hannah A , Otero Dennis C , Maganthi Swetha , Commisso Cosimo , Bradley Linda M
Published: August 2023

Background

Pancreatic ductal adenocarcinoma (PDAC) is highly refractory to all current immune checkpoint blockade (ICB) therapies and display limited CD8+ T cell infiltration. We previously showed that deficiency of P-selectin glycoprotein 1 (PSGL-1) promotes CD8+ T cell responses that clear chronic lymphocytic choriomeningitis virus (LCMV) Cl13 virus infection and dramatically inhibits the growth of anti-PD-1 blockade resistant melanoma. Here we evaluated if PSGL-1 regulates CD8+ T cell responses to PDAC tumors.

Results

PSGL-1−/− mice reproducibly showed T cell dependent, significant reduction of KPC.4662 primary PDAC tumors and reduced metastasis to lungs compared to control mice. Furthermore, anti-PD-1 treatment of PSGL-1−/− mice completely obliterated tumor growth as opposed to control mice. Histology of primary tumors revealed marked increase of immune infiltration (CD45+ cells), including CD3+ T cells in PSGL-1−/− mice. Flow cytometry and immunofluorescence staining confirmed a significant increase in tumor-infiltrating CD8+ T cells in PDAC tumors from PSGL-1−/− mice. Importantly, PSGL-1−/− CD8+ T cells were phenotypically and functionally less exhausted.

Conclusion

PSGL-1 deficiency significantly reduced growth of primary orthotopic PDAC tumors and decreased metastases and this effect was enhanced in combination with anti-PD-1 ICB treatment. This protective effect was associated with increased immune cell infiltration in the tumors and notably, increased CD8+ T cell effector responses. PSGL-1 therefore represents a novel target for promoting immune responses to PDAC tumors and responses to PD-1 blockade.

NCI Cancer Center Support Grant P30 CA030199
The Toll-like receptor 4 pathway modulates response to anti-PD-1 therapy
Authors: Whiteside Sarah K , Beresford-Jones Benjamin S , Sadar Puspendu , Kuo Paula , Xia Wangmingyu , Suyama Satoshi , Evans Alexander C , Imianowski Charlotte J , Conti Alberto G , Wesolowski Alexander J , Soderholm Amelia , Tourlomousis Panagiotis , Okkenhaug Klaus , Bryant Clare E , Pedicord Virginia , Roychoudhuri Rahul
Published: August 2023

Immune checkpoint inhibitors (ICI), such as anti-PD-1, have revolutionized cancer treatment. The use of antibiotics can lead to abnormal gut microbiome composition resulting in primary resistance to ICI. However, the mechanism by which this occurs is poorly understood. Metagenomic analyses of melanoma patient stool samples prior to therapy revealed an association between clinical responses to anti-PD-1 and functional differences in gut bacteria, including Gram-negative bacteria capable of lipopolysaccharide biosynthesis. By modeling anti-PD-1 therapy in mice, we found an antibiotic that binds lipopolysaccharide inhibited therapeutic efficacy. Subsequently, the inhibition of TLR4 signaling resulted in a loss of anti-PD-1 efficacy. Oral supplementation with lipopolysaccharide augmented anti-PD-1 immunology responses. These results reveal a TLR4-dependent role for gut-derived lipopolysaccharide in modulating anti-PD-1 responses.
Mitochondria targeted metabolic inhibitors promote lymphocyte infiltration into murine pancreatic tumors
Authors: Poimenidou Maria , AbuEid Mahmoud , Drouillard Donovan , McAllister Donna , Dwinell Michael B
Published: August 2023

Pancreatic ductal adenocarcinoma is a malignant carcinoma with a 5-year survival rate of 11% characterized with poor prognosis, rising incidence, and resistance to chemo- and immune-therapies. Cancer cell evasion and suppression of anti-tumor immune responses in pancreas cancer are regulated in part by metabolic reprogramming within cancer and tumor accessory cells. We sought to determine if targeted inhibition of mitochondrial oxidative phosphorylation by mito-metformin (MMe) and newly developed variant pCF3-MMe influence the tumor immune microenvironment. Our results showed that MMe strongly inhibited tumor growth in murine models of pancreas cancer. Flow cytometry revealed a significant increase in total CD45+ leukocytes, with elevated cytolytic granzyme B+ and CD44+ CD8+ T cells. Immunodepleting CD8+ T cells, reversed the anti-tumor effects of MMe. Immunofluorescence microscopy and immunoblot analysis indicated that MMe stimulated oligomerization of mitochondrial antiviral-signaling protein (MAVS) and phosphorylation of IRF3 and IRF7. Further, levels of the IRF3/7 target chemokines CXCL9, CXCL10, and CXCL11 were increased in MMe-treated tumor cells, consistent with a role in CD8+ T cell infiltration. These data indicate OXPHOS inhibition stimulated virus-independent MAVS oligomerization, activation of downstream proinflammatory signaling, and reignition of the suppressed pancreatic tumor immune microenvironment.

Funding Provided by NIH (2211376.1-5 R01 CA226279-01.04) and Center for Immunology MCW
Investigation of γδ T cell function in a novel inducible model of lung adenocarcinoma
Authors: Horrigan Katherine J , Lenahan Sean M , Mathieu Erin E , Moquin Cameron A , Dienz Oliver , Seward David J , Boyson Jonathan E
Published: August 2023

γδ T cells have been demonstrated to both promote tumor growth/metastasis and to efficiently kill tumor cells. This dual role of γδ T cells has been attributed to their ability to produce either IL-17 (γδT17) or IFNγ (γδT1), the former being associated with tumor progression and the latter being associated with anti-tumor activity. γδ T cell function is influenced by multiple factors, including the recognition of stress-induced ligands, as well as cytokines and chemokines. However, the specific signals that lead to the selective activation and function of γδT17 or γδT1 subsets in different cancers remains unclear. Here, we explored these questions using a novel inducible mouse model of Kras-driven lung adenocarcinoma with or without a co-mutation in Stk11, a master regulator of metabolic pathways. Preliminary characterization of this model revealed that KrasG12D/Stk11fl/fl mice exhibited significantly greater morbidity than KrasG12D mice. While lung leukocyte numbers did not differ significantly between KrasG12D and KrasG12D/Stk11fl/fl mice, we noted a significantly increased frequency of γδT17, but not γδT1, subsets in KrasG12D/Stk11fl/fl lungs. Interestingly, the increased frequency of γδT17 was due to increased Vγ1 and Vγ6, but not Vγ4, γδT17 suggesting a selective expansion of these subsets. To examine a potential role for butyrophilins in these models, we assessed lung butyrophilin gene expression using qPCR. This analysis revealed increased expression of Btnl1, Btnl2, and Btnl9 in KrasG12D lungs mice as compared to controls. Together, these results suggest that this model has the potential to shed new light on how the interaction of the tumor microenvironment with specific γδ T cell subsets affects disease pathogenesis.
PSGL-1 deficiency re-wires T cell signaling to support development of stem cell like exhausted T cells that control PD-1 blockade resistant melanoma tumor growth
Authors: Otero Dennis C , Hope Jennifer L , Palete Ashley B , Faso Hannah A , Lin Michelle , Roy Sreeja , Wang Eric S , Chow Savio , Yip Kevin , Campos Alexandre Rosa , Bradley Linda M
Published: August 2023

We previously identified that the adhesion molecule P-selectin glycoprotein ligand-1 (PSGL-1) regulates CD8+ T cell effector functions and promotes exhaustion in responses to chronic viral infection and melanoma tumors. In this study, we investigated cellular and molecular mechanisms by which intrinsic PSGL-1 signaling regulates the differentiation of exhausted T cells (TEX). We show that PSGL-1 localizes with the TCR upon co-ligation and find that PSGL-1 associates with Zap70 and the Zap70 inhibitor, Sts-1. In the absence of PSGL-1, expression of Sts-1 is reduced allowing for greater activation of Zap70 and the downstream signaling molecules Akt and Erk1/2. In addition, PSGL-1-deficiency empowers CD8+ T cells to respond to low affinity TCR ligands. In vivo, PSGL-1 deficiency leads to growth inhibition of a PD-1-blockade resistant melanoma. Notably, PSGL-1-deficiency in T cells enables retention of effector T cell (TEFF) functions, and an increased representation of stem cell-like T cells (TSC) that express TCF-1 and low levels of TOX, which are associated with sustained effector responses and responsiveness to ICB. We now show that concomitant PSGL-1 signaling with repeated TCR stimulation of human TEFF elicits functional exhaustion. Importantly, blockade of PSGL-1 recapitulates PSGL-1 deficiency in anti-tumor as well as antiviral responses, and its distinct mechanism of T cell inhibition underscores the translational potential of targeting PSGL-1 by ICB.

Supported by grants from NIH (R01 AI106895, R21 CA249353, R21 CA216678, R03 CA252144) Melanoma Research Alliance MRA 696326, Department of Defense W81XWH-20-1-0324, Sanford Burnham Prebys NCI-Designated Cancer Center Support Grant, P30 CA030199 American Cancer Society Postdoctoral Fellowship (PF-20-113-01-LIB)
Human CAR19 CD8+ iTreg exceed CAR19 CD8 CTLs both in suppressing GVHD lethality and eliminating CD19+ Nalm-6 lymphoma cells in vivo
Authors: Larson Jemma H , Compeer Ewoud B , Torghabeh Mehrdad Hefazi , Smith Kyle D , Jin Sujeong , Dougherty Phillip , McDonald-Hyman Cameron , Houle Bailey , Dustin Michael , Hippen Keli L , Blazar Bruce R
Published: August 2023

Simultaneously achieving a graft versus leukemia (GVL) effect while suppressing graft versus host disease (GVHD) would facilitate the development of immunotherapies for hematopoietic stem cell transplantation. Here, we show that human peripheral blood CD8+CD25− T-cells cultured with IL-2, TGF-β and rapamycin generates CD8 induced Tregs (iTregs). CD8 iTregs expressed FoxP3, CD25 and higher CD103 and CD39, but lower Helios, CTLA-4, and TIGIT than CD4 Tregs. While in vitro suppressor function was modestly reduced with CD8 iTreg, in vivo suppression of GVHD lethality was comparable to that of CD4 Treg. In vitro, cytolytic activity with an αCD3×αCD19 bispecific engager against CD19+ Nalm-6 lymphoma cells was comparable to CD8 CTLs. We subsequently generated CD8 iTreg and CTLs expressing αCD19scFv (CAR19). Following CAR19-EGFR sort purification, CAR19 CD8 iTreg and CAR19 CTL had similar expression of perforin, IFNγ and TNFα. In a 48h Incucyte killing assay against CD19+Nalm-6 targets, using CAR19 CD8 iTreg and CAR19 CTLs generated from the same donors, no significant differences were noted; CD8 iTreg killing was perforin-dependent and granzyme-independent. No killing was observed against CD19 koNalm-6 targets. Unexpectedly, CAR19 CD8 iTreg were found to be significantly more effective at limiting tumor burden in vivo and significantly delayed tumor-related mortality in a xenogeneic Nalm-6 tumor model compared to CAR19 CTLs (p = 0.0018). Mechanistic studies are ongoing. Together, these findings highlight distinct advantages of CD8 iTreg as a concurrent immunotherapy and provide a rationale for CD8 iTreg based CAR therapies to prevent GVHD and promote GVL.

Supported by grants from NIH (R01 HL11879-07 and 2P01 CA065493-25) as well as funding from the Childrens’ Cancer Research Fund
Tissue Engineering Scaffolds Remotely Surveil Presymptomatic Allograft Rejection
Authors: Urie Russell R , Xiao Chengchuan , Hughes Elizabeth , Lombard Elizabeth , Chen Judy , Morris Aaron , Farris Diana , Goldstein Daniel , Shea Lonnie
Published: August 2023

As there is no assay to predict the risk of acute cellular allograft rejection (ACAR), clinicians rely on graft biopsy and aggressive, one-size-fits-all immunosuppression. Endomyocardial biopsy (EMB) is a flawed standard for heart transplant surveillance and diagnosing ACAR, as histological evidence of rejection inherently lags behind molecular biomarkers and suffers from variability. Noninvasive alternatives to EMB in ACAR surveillance, including gene profiling and donor-derived cell-free DNA, also measure lagging indicators of ACAR. A minimally invasive surveillance method is urgently needed to quantify early risk of rejection for minimizing biopsy and personalizing immunosuppression. We have developed porous biomaterial “scaffolds” for minimally-invasive sampling. These scaffolds amass immune cells producing biomarkers as an immunological niche.

We identified predictive biomarkers of ACAR conserved in both in murine heterotopic heart transplant and skin transplant through the remote scaffold. We identified 43 highly differentially-expressed genes as organ-agnostic biomarkers of T cell-mediated rejection. These novel biomarkers differentiate between pre-symptomatic ACAR and recipients with healthy allografts, without invasive biopsy. Scaffold cell capture technology provides a leading indicator of ACAR by monitoring tissue-specific immune responses associated with allograft rejection. This implantable scaffold enables minimally invasive histological evaluation and molecular calculation of the early risk of rejection to reduce the frequency of routine EMB and personalize immune suppression that could prolong transplant life while minimizing risk.

Supported by the Falk Medical Research Trust Catalyst Award and Transformational Award
TLR2 in γδ T cells is required for metabolic support of their IL-17A response to a commensal bacterium on the ocular surface
Authors: Xu Xiaoyan , Zhu Wenjie , Peng Zixuan , Caspi Rachel
Published: August 2023

The ocular surface is colonized by commensal microbiota, which tune the local mucosal immune response. We previously identified an ocular surface commensal, Corynebacterium mastitidis (C. mast), which induced IL-17A production from γδ T cells in the conjunctiva and eye-draining lymph nodes (DLN), and enhanced host resistance to fungal and bacterial pathogens. To elucidate how IL-17A producing γδ T cells (γδT17) are activated by C. mast, we associated WT and TLR2−/− mice with C. mast, and investigated the role of TLR2 in this process. This was based on the rationale that C. mast is a Gram-positive bacterium and that TLR2 expression on γδT cells increased after C. mast association. Mechanistic studies showed that TLR2 expression was required not only on DCs (for IL-1 production) but also on the γδT cells. RNA-Seq revealed that genes related to mitochondrial oxidative phosphorylation were dysregulated in TLR2−/− γδT17 cells, indicating that engagement of TLR2 may regulate metabolic processes in γδT17 cells. Seahorse assay confirmed that TLR2−/− γδ T17 cells displayed impaired oxygen consumption rates and lower ATP production. Interestingly, lysosomal activity, which plays a central role in cellular metabolism, was decreased in TLR2−/− γδ T17 cells. Enhancement of lysosomal activity by curcumin C1 restored ATP generation in TLR2−/− γδ T17 cells. We propose that intrinsic TLR2 signaling in γδ T cells is required for metabolic support of their IL-17A response.
Sympathetic regulation of skin microbiota-induced innate-like T cells
Authors: Rao Indira N , Enamorado Michel , Merrill Dean , Link Verena , Gribonika Inta , Bouladoux Nicolas , Belkaid Yasmine
Published: August 2023

Recent discoveries in microbiota-mediated neuroimmune crosstalk highlight how co-evolved systems collaboratively facilitate host fitness. However, in the skin – the largest and outermost protective barrier – little is known about the role of microbiota in neuro-immune regulation. We find that murine skin colonization with commensal Staphylococci induces significant accumulation of dermal IL-17A-producing Vg6+ gdT (Vg6 gdT17) cells in IL-1 and microbiota-dependent manner. Compared to sedentary Vg6 gdT17 from unassociated controls, intravital imaging shows that commensal-induced dermal Vg6 gdT17 form large highly motile aggregates. Transcriptomic analysis revealed significant differences between Vg6 gdT17 from commensal-colonized mice versus unassociated controls. Among these, the cells from colonized mice showed significant downregulation of sympathetic beta-adrenoreceptor (bAR) expression. Beta-AR signaling has recently been shown to impact conventional T cell motility in the skin. We observe that, upon systemic b2AR agonist administration, the highly dynamic commensal-induced dermal Vg6+ gdT17 clusters are rendered immobile, showing sympathetic control of gdT17 cell motility. Intriguingly, skin commensal exposure leads to systemic induction of Vg6+ gdT17 cells, implying a role for these cells beyond local surveillance. Further studies are underway to test whether, following exposure to skin commensal, sympathetic regulation promotes inter-organ gdT cell dissemination and impacts their functional potential. Overall, this work aims to uncover the mechanism(s) underlying microbiota-induced skin neuroimmune interactions in promoting organismal homeostasis.
Pathogenesis of flares in liver inflammation in hepatitis B virus-infected women during and after pregnancy
Authors: Fukutomi Keisuke , Nishio Akira , Hasan Sharika , Shah Pir A , Ali Mukarram J , Campoverde Reyes Karen J , Badger Jonathan H , Trinchieri Giorgio , Lau Daryl T Y , Rehermann Barbara
Published: August 2023

Women with chronic hepatitis B (CHB) are at risk of increased liver inflammation during pregnancy. To investigate the pathogenesis of these hepatic flares we studied immune responses and gut microbiota in 19 women with CHB (12 without and 7 with antiviral (NUC) treatment) and 3 uninfected controls in each trimester and at two and six months postpartum. Increased serum alanine aminotransferase (ALT) activity as a liver inflammation marker was defined as a value greater than the upper limit of normal and twice the baseline value.

While increased plasma levels of soluble CD163, inflammatory cytokines and chemokines generally occurred during episodes of liver inflammation, increases of IL-22 and IFN-γ levels during pregnancy preceded a postpartum ALT increase. In contrast, levels of IL-27, a cytokine produced by the trophoblast, increased also in women with normal ALT levels.

The fecal microbiota of HBV-infected patients with and without NUC treatment had lower α-diversity (inverse Simpson index) than those of uninfected controls. 16srRNA amplicon analysis and PCA showed patient-specific clustering of microbiota. To identify factors predictive of a postpartum ALT increase, we compared 16srRNA DNA data from patients with a postpartum ALT increase to those with normal postpartum ALT levels (four patients per group). Combining all time points, the gut microbiota of those with postpartum ALT increase differed significantly from those with normal ALT activities (UMAP, PERMANOVA p<0.003).

In conclusion, the composition of the fecal microbiota was patient-rather than time point-specific and differed between patients with and without postpartum ALT increase.

Supported by NIH Bench-to-Bedside Award (B.R. and D.T.Y.L.)
Shedding LIGHT and TL1A on Pulmonary Fibrosis
Authors: Steele Hope , Cheng Jason Liang , Willicut Ashley , Dell Garrison , Breckenridge Joey , Ghastine Andrew , Herro Rana
Published: August 2023

Currently, anti-inflammatory drugs fail to reduce pulmonary fibrosis (PF) and tissue remodeling in the clinic. Despite the advancements made in understanding the molecular pathways driving PF, there is still an unmet need to develop innovative and effective anti-fibrotic drugs. Our lab has identified two novel mediators of fibrosis belonging to the tumor necrosis factor superfamily (TNFSF), LIGHT (TNFSF14) and TL1A (TNFSF15). Using a well characterized mouse model of PF driven by bleomycin, we showed that blocking LIGHT or TL1A signaling in isolation, by genetic ablation or antibody-mediated neutralization, significantly reduces PF. In gain of function studies, the in vivo administration of LIGHT or TL1A in isolation recapitulated fibrosis symptoms reminiscent of human disease. Furthermore, our data suggest that LIGHT promotes thymic stromal lymphopoietin (TLSP) expression by epithelial cells, whereas TL1A activates fibroblasts to promote periostin expression. Both TSLP and periostin are drivers of PF. Importantly, we observed an upregulation of LIGHT and DR3 (the receptor for TL1A) in human lung biopsies of patients suffering SSc and IPF. We have found that LIGHT and TL1A can drive fibrosis independently of one another and that the in vivo co-administration of these TNFSF members synergize to maximize inflammation and collagen deposition. Using antagonistic fusion proteins to neutralize LIGHT and TL1A signaling, we will monitor PF in vivo and check whether greater efficacy is achieved when LIGHT and TL1A are targeted concomitantly post-disease onset. The relevance of this work on PF associated with ILDs in humans is tremendous.
Improving lymphatic flow in SLE mice induces fibroblastic reticular cell survival via lymphotoxin beta receptor signaling
Authors: Howlader Mir , Lu Theresa
Published: August 2023

Systemic lupus erythematosus (SLE) patients are photosensitive, where ultraviolet radiation (UVR) exposure can induce skin inflammation and also trigger systemic disease flares. The link between photosensitive skin and systemic autoimmunity is not well understood. Skin communicates with the immune system via lymphatic vessels that carry lymph into the conduit system of lymph nodes (LNs), which is ensheathed by fibroblastic reticular cells (FRCs). FRCs play critical roles in limiting T and B cell responses. Dendritic cells (DCs) interact both with lymphatic fluid and with FRCs and can modulate FRC survival and numbers via a DC-lymphotoxin beta receptor (LTβR)-FRC axis. We have recently shown dysfunctional lymphatic flow in human SLE and SLE mouse models exposed to UVR and that improving lymphatic flow via manual lymphatic drainage (MLD) reduces skin photosensitivity and LN plasmablast responses, while increasing FRC numbers. In this study, we aim to delineate the mechanism by which MLD induces FRC alterations, hypothesizing that it is via the DC-LTβR-FRC axis. Currently, we have found that MLD increases FRC survival of UVR exposed mice. However, treatment with antagonist LTbR-Ig reverses this FRC survival, suggesting that MLD induces FRC survival via LTbR signaling. Ongoing experiments are targeting the importance of DCs and DC-derived LTβ in modulating MLD-induced changes to FRCs. The novelty of this study is in showing that lymphatic flow from the skin can influence stromal cell populations in SLE. These findings will improve our understanding of the relationship between photosensitivity and systemic autoimmunity.
Microenvironment regulates antibody secretion by IgA+ plasma cells
Authors: Zoccoli-Rodriguez Victoria E , Coombs Wanda , Harris Joshua , Delaney Mara , Wilmore Joel
Published: August 2023

IgA secreting plasma cells (PCs) provide a critical supply of antibodies for barrier functions against pathogens. These cells express unique tissue-driven transcriptional profiles in their respective niches in the bone marrow (BM) and gut. We previously demonstrated that both tissues sustain long-lived IgA+ PCs, but the cell intrinsic and extrinsic signals responsible for survival and function in the gut remain largely unknown. Understanding how the microenvironment regulates tissue resident IgA+ PC functions has implications for maintaining intestinal homeostasis and protection from disease. We found that BM IgA+ PCs functionally differ from gut IgA+ PCs as they secrete significantly more antibody. To assess if this phenotype could be due to metabolic consequences, we profiled IgA+ PCs in both tissues. BM and gut IgA+ PCs have similar glucose and fatty acid uptake. However, BM IgA+ PCs have increased mitochondrial dependence, while gut IgA+ PCs have increased glycolytic capacity. We next altered the composition of the microbiome to determine the role of gut microbes in regulating tissue resident PCs. Our initial experiments used C57BL/6J mice with a well-defined low-complexity microbiome (from Jackson Labs). Co-housing Jax mice with animals containing a more diverse set of microbes led to an increase in antibody secretion. Herein, we identified that the tissue microenvironment and gut microbiome influences PC function. We aim to dissect the mechanisms involved in microbiome driven changes in PC phenotype. Ultimately, understanding how the microenvironment regulates intensity of PC antibody secretion provides a tool in targeting induction of systemic and mucosal responses and will improve vaccine design and development.

Supported by grants from NIH (R35 GM147560, R03 AI156481)
Kinetics of endogenous thymic regeneration following chemotherapy-induced involution
Authors: Lagou Maria K , Carpenter Randall S , Anastasiadou Dimitra P , Hardas Alexandros , Garcia Raquel Vallejo , Argyris Dimitris G , Chondronikola Anastasia-Aristoteleia , Licausi Francesca , Briceno Andrea , Guzik Hillary , DesMarais Vera , Branch Craig A , Cui Min-Hui , Poutahidis Theofilos , Angelopoulou Katerina , Maryanovich Maria , Karagiannis George S
Published: August 2023

The thymus is a primary lymphoid organ where T cell development occurs. Besides the naturally transpiring age-dependent involution, the thymus also undergoes acute involution from cytotoxic stimuli. Establishing thymic regeneration strategies would, thus, be immensely critical for pediatric cancer and transplantation patients receiving cytoablative chemotherapies. Several signaling pathways (e.g., KGF, BMP4) promote thymic regeneration, albeit with suboptimal therapeutic potential, mainly due to poor insight of the detailed mechanisms of endogenous thymic repair. To address this issue, we developed a mouse model of cyclophosphamide-induced thymic involution and monitored thymic reconstitution for up to 5 weeks post-treatment. Macroscopic assessment via thymic weight index and MRI-based volumetric analysis, along with histopathology, revealed that endogenous thymic regeneration is a non-linear, multistep process, consisting of a: (a) Reconstitution phase, during which recuperation occurs at an exponential rate, (b) Rebound phase, in which abnormal thymic hyperplasia is observed, and (c) Recoil phase, where thymic homeostasis ensues. Quantification of peripheral blood lymphocytes, however, revealed that thymic hyperplasia at Rebound does not correlate with restored thymic functions and output. Detailed investigation via flow cytometry revealed the stalling of T cell development at the Double Positive (CD4+ CD8+) stage, implying cortical defects during thymic repair. Currently, we employ single-cell RNA sequencing with single cell resolution imaging, to define the molecular pathways disrupting cortical Thymic Epithelial Cell (cTEC) renewal post-chemotherapy, leading to impaired thymopoiesis.

Supported by AAI’s Career in Immunology Fellowship PI’s funding supported by Albert Einstein College of Medicine: New Investigator Start-up funds
In vitro validation of Cathepsin S docking models
Authors: DeHority Riley , Gil Pineda Laura I , Cochran Kari , Bian Yuanzhi , Lemkul Justin A , Zhang Chenming
Published: August 2023

In dendritic cells, the dominant lysosomal protease involved in Ii chain degradation and antigen digestion is Cathepsin S, a cysteine protease in the papain family. Extracellularly, Cathepsin S also contributes to pathological processes in cardiovascular disease, cancer, and autoimmune arthritis: unlike most lysosomal proteases, it retains its activity at serum pH. In computational molecular docking studies comparing peptides from the literature, we found that Cathepsin S shows a pH-dependent specificity switch, with predicted dissociation constants for specific peptide sequences differing at lysosomal and serum pH. The aim of this study is to further characterize this specificity switch and to compare docking results to kinetic parameters of Cathepsin S digestion in vitro. We identified peptide sequences of interest from docking, including sequences from the Ii chain and elastin, and digested these peptides (LifeTein) using human Cathepsin S (Calbiochem), collecting samples for LC-MS analysis over 16 hour reaction times. We report the kinetic parameters of Cathepsin S catalysis at both lysosomal and serum pH. Digestion results are compared to docking models and the models most predictive of catalytic activity are identified. Further investigation is needed to determine whether pH-dependent catalytic activity and selectivity of Cathepsin S will enable the design of protein-based therapies and inhibitors optimized for their targeted microenvironments.

This work was partially supported by the NIDA/NIH award UG3DA048775, the USDA National Institute of Food and Agriculture, AFRI project #2021-0858, and the department of Biological Systems Engineering at Virginia Tech.
Mechanistic aspects of tapasin-mediated antigen presentation revealed by structure of a tapasin/MHC-I complex
Authors: Natarajan Kannan , Jiang Jiansheng , Taylor Daniel Kyle , Kim Ellen J , Boyd Lisa F , Dhobi Javeed Ahmad , Mage Michael G , Truong Hau , Woodward Claire , Sgourakis Nikolaos , Cresswell Peter , Margulies David H
Published: August 2023

T cell immunity to viruses and cancers is crucially dependent on the cell surface display of major histocompatibility complex class I (MHC-I) molecules selectively loaded with high affinity peptides. Peptide loading occurs in the ER within a multimolecular assembly known as the peptide loading complex (PLC). A critical component of the PLC is tapasin, a 45 kDa membrane glycoprotein in whose absence the expression and stability of MHC-I are greatly reduced. To determine the mechanistic basis of tapasin-mediated peptide loading, we determined the crystal structures of human tapasin in complex with HLA-B44:05 as well as tapasin complexed to each of two well-characterized antibodies. The tapasin-stabilized peptide receptive state is characterized by distortions of the peptide binding groove, destabilization of the b2-microglobulin interaction, and rearrangements of the membrane proximal Ig domains, all of which are reversed on high affinity peptide binding. Additionally, the structural footprints of the anti-tapasin antibodies confirm previous functional assays and together with the tapasin/MHC-I complex and mutagenesis data reveal dynamic aspects of tapasin function.

Supported by the Intramural Research Program, NIAID, NIH.
Cell-specific MHC class I deletion on CD11c+ APCs abrogates CD8 T Cell infiltration into the brain
Authors: Maynes Mark Andrew , Fain Cori E , Ayasoufi Katayoun , Wolf Delaney , Tritz Zachariah , Bamkole Michael A , Jin Fang , Hansen Michael J , Johnson Aaron J
Published: August 2023

Cerebral malaria (CM) is a severe complication of P. falciparum infection and a leading cause of death, globally. Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice serves as a murine model of CM called experimental cerebral malaria (ECM) due to the neurological and neuropathological features that simulate the infection of human CM. A putative mechanism of disease in human CM requires CD8 T cell to infiltration into the brain and blood-brain barrier disruption. The specific antigen-presenting cell type(s) that prime this CNS infiltrating CD8 T cell response capable of inducing lethal blood-brain barrier disruption has not been defined. To answer this, single class I expressing Kb loxP transgenic mice were generated. Crossing these mice to class I deficient CD11c-cre or LysM-cre mice created the LysM-cre Kb cKO and CD11c-cre Kb cKO mice, respectively. PbA infected Kb loxP and LysM-cre Kb cKO were susceptible to ECM. In contrast, CD11c-cre Kb cKO mice survived acute PbA infection and avoided neurological manifestations. We therefore employed spectral flow cytometry to analyze splenocytes and brain infiltrating immune cells during acute PbA infection in these genotypes. We observed a reduction in activated CD8 T cells in the spleens of PbA infected CD11c-cre Kb cKO mice versus the Kb loxP mice and the LysM-cre Kb cKO. This reduced peripheral CD8 T cell activation in PbA infected CD11c-cre Kb cKO mice also corresponded with reduced brain infiltration of CD8 T cells. We conclude a CD11c+ APC is therefore required for priming and potentially recruitment of CD8 T cells to the brain during lethal ECM pathology.

5R01NS103212-06
Xeno-interactions between MHC-I proteins and molecular chaperones enable ligand exchange on a broad repertoire of HLA allotypes
Authors: Sun Yi , Papadaki Georgia , Devlin Christine , Danon Julia , Young Michael , Winters Trenton , Burslem George , Procko Erik , Sgourakis Nikolaos
Published: August 2023

Immunological chaperones Tapasin and TAPBPR play key roles in antigenic peptide optimization and quality control of nascent MHC-I molecules. The polymorphic nature of MHC-I proteins leads to a range of allelic dependencies on chaperones for assembly and cell-surface expression, limiting chaperone-mediated peptide exchange to a restricted set of human HLA allotypes. Here, we demonstrate and characterize xeno-interactions between a chicken TAPBPR ortholog and a complementary repertoire of HLA allotypes, relative to its human counterpart. We find that TAPBPR orthologs recognize empty MHC-I with broader allele specificity and facilitate peptide exchange by maintaining a reservoir of receptive molecules. Deep mutational scanning of human TAPBPR further identifies gain-of-function mutants, resembling the chicken sequence, which can enhance HLA-A* 01:01 expression in situ and promote peptide exchange in vitro. These results highlight that polymorphic sites on MHC-I and chaperone surfaces can be engineered to manipulate their interactions, enabling chaperone-mediated peptide exchange on disease-relevant HLA alleles.

This work was supported by NIAID (5R01AI143997), NIDDK (5U01DK112217), NIGMS (5R35GM125034), and NIGMS (R35GM142505).
Hexavalent coronavirus vaccine elicits both humoral and cellular immune responses in mice
Authors: Gröhn Stina , Soppela Saana , Iakubovskaia Alina , Pasternack Arja , Lampinen Vili , Ritvos Olli , Hankaniemi Minna
Published: August 2023

Coronaviruses have caused three deadly human outbreaks over the last 20 years, the latest causing the COVID-19 pandemic which has now lasted over three years. The animal reservoir of coronaviruses is extensive, and history has shown that coronaviruses are able to crossover from animals to humans. Thus, there is a clear need for a universal coronavirus vaccine. In this study, we produced S1 subunits of spike proteins of six different human coronaviruses fused to the antibody Fc fragment in HEK293 cells. In addition, we produced another coronavirus protein (CP) in High Five™ insect cells. The vaccine compositions included either the six S1-Fc or the six S1-Fc together with CP, and mice were immunized either intranasally or with a combination method utilizing both subcutaneous and intranasal injections. Our results showed that both vaccine compositions elicited both humoral and cellular immune responses in mice. Antigen-specific IgG levels were higher in mice immunized solely intranasally but using a combination of different immunization routes seemed to boost cellular immune responses. As a conclusion, all the vaccine compositions were highly immunogenic and generated both antibody and T cell responses in mice. Further studies are needed to optimize the immunization scheme. Additionally in the future, in order to better characterize the T cell responses generated by intranasal immunization, the nasal associated lymphoid tissue and cervical lymph nodes from the mice will be collected and analyzed.

Supported by a grant from The Research Foundation of the Pulmonary Diseases (Finland)
The role of intestinal intraepithelial lymphocytes in resistance against coccidiosis in chickens
Authors: Nazmi Ali , Hamad Shaimaa K , Majeed Shuja
Published: August 2023

Coccidiosis is a critical enteric disease that costs more than $14.5 billion in annual cost to world poultry industry. It is caused by different Eimeria species that invade and replicate within the intestinal epithelium in a site-specific manner. The Eimeria infection induces local intestinal damage, reduces feed consumption, feed utilization, and growth performance, and increasing mortality rate. With the recent antibiotic ban including anticoccidial drugs in chicken production, the live vaccination become widely used to control coccidiosis. However, the live vaccines are expensive, require time to circulate in the host to induce a protective immunity, and process a high-risk factor for necrotic enteritis caused by clostridium perfringens. Therefore, there is a high demand for development of safe, protective, and inexpensive vaccines against coccidiosis which require better understanding of host immune responses to Eimeria parasites. The aim of this study is to elucidate the protective role of intestinal intraepithelial lymphocytes (IELs) during the Eimeria infection. At 3 week of age, SPF white-leghorn chicks were divided into 3 groups. Each group (n=50) was orally inoculated with 20,000 oocytes of Eimeria (E) acervuline or E. maxima, or mock-inoculated with PBS. At 2 day post-infection (dpi), 7 dpi and 14 dpi, the frequency and the number of small intestinal IEL populations were determined by a flow cytometry. Eimeria infection induced the number of TCRγδ+, TCRβ+, TCRneg, TCRβ+CD4+, TCRβ+CD4+CD8α+, TCRβ+CD8αβ+, TCRβ+CD8αα+, and iCD8α IELs compared to the control group at 14 dpi. However, there was no difference among groups at 2 and 7 dpi. More experiments are underway to determine the effector and function of each subset of IELs.

Startup funding, The OSU
Human BH3-only protein, Noxa, is a major contributor to proliferative metabolism and apoptosis in the CD8+T cell immune response
Authors: Yang Tingyuan , Sharma Himani , Pennell Christopher A , Kelekar Ameeta
Published: August 2023

Human Noxa, the smallest BH3-only Bcl-2 protein, was originally identified as pro-apoptotic, but has since also been shown to promote cell growth in both malignant and activated normal T cells by facilitating proliferative metabolism (Lowman et al., 2010, Mol Cell, 40, 823–33; Hanse et al., 2017, Oncogene, 36, 3915–3924). To investigate the contribution of Noxa to the T cell immune response, we tracked its expression in primary human CD8+T cells in an in vitro anti-CD3/CD28 co-stimulation model. Noxa is undetectable in naïve CD8+T cells but is induced upon co-stimulation and remains highly expressed during the differentiation phase and through the onset of apoptosis. The viable cells that remain following apoptosis at the end of the immune response are ‘memory-like’, as determined by flow cytometry, harboring large mitochondria with robust spare respiratory capacity. These cells no longer express Noxa, but rapidly upregulate it upon re-stimulation. We observe that Noxa protein expression in CD8+T cells, upon stimulation, is directly dependent on glutamine and unaffected by glutaminase inhibitors, while the entry of glutamine carbons into the TCA cycle, in turn, requires Noxa. CD8+T Noxa KO cells, moreover, show reduced dependence on glutamine and significantly higher viability at the end of the immune response. CD8+T cells expressing an apoptosis deficient Noxa BH3 domain point mutant similarly presented an extended effector phase. The fundamental contributions of human Noxa to both activation-associated metabolic reprogramming and apoptosis in a prototypic immune response, makes this protein an attractive tool to manipulate the expansion, effector function, and longevity of CD8+T cells for immunotherapy.

R21 AI148876