bluuebunny/crossref_metadata_2025 huggingface dataset:
Deletion of both Id2 and Id3 in developing T cell promotes NKT lymphoma formation in mice (TUM2P.887)
Authors: Li Jia , Jima Dereje , Dave Sandeep , Zhuang Yuan
Published: January 2023

NKT lymphoma is a rare form of tumor, the etiology and pathogenesis of which is still largely unknown. There are also no animal models to study its course. The Id2 and Id3 genes and their targets, E proteins, have been demonstrated to be involved in multiple lymphoma and cancer subtypes, but their role in the development of NKT lymphomas is unexplored. Here we show that combined deletion of Id2 and Id3 by LckCre in early T cell development results in NKT cell expansion, which rapidly progresses into malignancy. Adoptive transfer experiments indicate that the malignant cells are able to invade healthy tissue. cDNA Microarray analysis of NKT lymphoma and pre-malignant NKT cells showed several cytokine signaling pathways are altered during tumor progression. Therefore, regulation of E proteins by Id2 and Id3 may play a role in NKT lymphoma development. Furthermore, our double knockout mice may provide a model to study mechanisms involved in human NKT lymphoma progression.
The change of T cell repertoire due to central tolerance determines the antigenic requirement and strategy for inducing effective anti-tumor CD8 T cell response (TUM2P.888)
Authors: Leng Qibin , Guo Nining , Zhu Kai , Zhang Na , Yang Chunhui , Wang Jianrong , Yang Juhao , Luo Kaimin , Wang Yan , Shen Hao , Zehn Dietmar , Leng Qibin
Published: January 2023

Tumor antigens are mainly low-avidity antigens to T cells and unable to elicit effective anti-tumor immunity. Little knowledge on how low-avidity antigens elicit T cell response in vivo further hinders designing strategies to improve anti-tumor immune response. Using a unique mouse tumor model, we found that neither native tumor antigen nor majority of altered peptide ligands (APLs) derived from tumor antigen but only intermediate avidity APL was able to elicit robust cross-reactive tumor-specific CD8 T cell response. Central tolerance shifted T cell repertoire to allow host T cells only respond to low avidity APLs and spare the lower cross-reactivity to self antigens (tumor antigens). In addition, the functional avidity of cross-reactive tumor-specific CD8 T cells was over 1000 times less than that of pathogen-type antigen-specific CD8 T cells. These results indicate that the extremely low functional avidity and low cross-reactivity to native tumor antigen are two key reasons why APLs are ineffective for eliciting cytotoxic activity of CD8 T cells to kill tumor cells. Our further studies revealed that two distinct mechanisms control the functionality and responsiveness of tumor-specific CD8 T cells. This study also discovered a novel immunization strategy to enhance the functionality and responsiveness of tumor-specific CD8 T cells. Altogether, our study will shed light on strategies to enhance the efficacy of both immunotherapy and vaccination against cancers.
Neuropilin-1 enforces Treg stability and function in the tumor microenvironment. (TUM2P.889)
Authors: Overacre Abigail , Delgoffe Greg , Vignali Dario
Published: January 2023

Regulatory T cells (Tregs) play an integral role in the adaptive immune system by suppressing detrimental self-reactive responses. However, Tregs have deleterious effects in cancer through suppression of the anti-tumor immune response. Therefore, it is advantageous to understand the role of Tregs in the tumor microenvironment in order to create targeted cancer therapies. Our lab has uncovered a novel function for neuropilin-1 (Nrp1) on Tregs in tumors. In the absence of Nrp1 ligation by its ligand, Sema4a, Tregs lose stability and suppressive capabilities, leading to enhanced anti-tumor immunity and reduced tumor growth or tumor clearance. We hypothesize that the loss of Nrp1 expression on Tregs reduces intratumoral stability through altered intracellular pathways and diminished Foxp3 expression, transforming Tregs from an anti-inflammatory to a pro-inflammatory T cell population. Nrp1-deficient Tregs have a hypermethylated Foxp3 CNS2/TSDR locus in the tumor microenvironment, comparable to Teff, and display TH1-like characteristics, with upregulation of Tbet, CXCR3, and IFNγ, and the reduction of suppressive molecules, IL-10 and CD73. Furthermore, in competitive environments Nrp1-deficient Tregs undermine and limit the function of wild-type Tregs. Further mechanistic analysis and fate mapping studies will elucidate the cell-intrinsic and cell-extrinsic impact of Nrp1 loss on Treg function and tumor clearance.
Gene related to anergy in lymphocytes deficiency confers spontaneous tumor regression (TUM2P.897)
Authors: Wang Junmei , Zou Qiang , Sahoo Anupama , Alekseev Andrei , Sun Shao-cong , Nurieva Roza
Published: January 2023

T cell tolerance is the main obstacle to successful cancer immunotherapy; thus, today it is a high priority to develop strategies to break T cell tolerance and thereafter enhance T-cell cytotoxicity towards tumor cells. We, as well as other groups, have acknowledged the E3 ubiquitin ligase, Gene related to anergy in lymphocytes (Grail), as an essential component of T-cell tolerance phenotype. Here we report that Grail expression is highly upregulated in CD8+ T cells infiltrated into the EG-7 tumors compared to T cells in lymphoid tissues, and Grail deficiency confers spontaneous protection against inoculated EG-7 tumors. Importantly, therapeutic transfer of naïve tumor-antigen-specific Grail deficient CD8+ T cells into the wild-type hosts was sufficient to reject established tumors. Mechanistically, Grail deficient CD8+ T cells exhibit augmented effector cytokine expression and enhanced cytolytic function compared to wild-type CD8+ T cells, and are resistant to the suppression mediated by regulatory T cells. Moreover, the elevated cytotoxic function of Grail-deficient CD8+ T cells essentially depends on TCR and IL-21-signaling pathway; Grail promotes IL21R ubiquitination and degradation. Altogether, our data demonstrate that Grail is a crucial factor that controls anti-tumor activity of cytotoxic T cells, thus, inhibition of Grail may be a beneficial immunotherapeutic approach to induce potent immune responses against established tumors.
TCR transgenic and patient-derived CTL killing of CTL-resistant melanoma: modulation of TRAIL apoptotic pathway and immune sensitization by epigenetic modifier SAHA (TUM2P.901)
Authors: Jazirehi Ali , Kurdistani Siavash , Economou James
Published: January 2023

Resistance to apoptotic signals delivered by cytotoxic T cells is likely to become an important limiting factor with increasingly effective immune therapies. We report that TCR transgenic CTLs primarily use the TRAIL apoptotic pathway, via DR5 receptor, in killing sensitive melanomas. CTL-resistant melanomas had both DR5 down-regulation and aberrant expression of apoptotic molecules. Clinically achievable concentration of FDA approved chromatin remodeling drug SAHA increased surface DR5 expression, favorably inverted the expression of apoptotic genes, and restored the sensitivity of R melanomas to MART CTL. TCR transgenic MART CTLs were used as a reliable and reproducible source of highly potent melanoma-reactive CTLs. Acquisition of CTL-resistance and its reversal by SAHA was also confirmed using naturally occurring T cells expressing endogenous MART-1 TCR derived from melanoma patients. Further, using rhTRAIL and Drozitumab explicitly verified the contribution of DR5/TRAIL pathway. Apoptosis negative (c-IAP-2 and Bcl-xL) and positive (DR5) regulators were potential candidates partly regulating CTL sensitivity. Combination of SAHA and rhTRAIL or Drozitumab destabilized mitochondria and facilitated cytosolic release of apoptotic molecules. These preclinical findings suggest that exposure to chromatin remodeling drug of immune-resistant melanomas can skew towards an intracellular pro-apoptotic milieu, increase death receptor expression, and overcome acquired immune-resistance.
IP3 3-kinase B controls hematopoietic stem cell homeostasis and prevents lethal hematopoietic failure in mice (HEM5P.228)
Authors: Sauer Karsten , Conche Claire , Broaten Blake , Schaffer Lana , Rigaud Stephanie , Westernberg Luise , Head Steven , Siegemund Sabine
Published: January 2023

Tight regulation of HSC homeostasis ensures life-long hematopoiesis and prevents blood cancers. The mechanisms balancing HSC quiescence with expansion and differentiation into hematopoietic progenitors are incompletely understood. Here, we identify inositoltrisphosphate (IP3) 3-kinase B (Itpkb) as a novel essential regulator of HSC quiescence and function. Young Itpkb-/- mice accumulated phenotypic HSC which were less quiescent and proliferated more than wildtype controls. Itpkb-/- HSC downregulated quiescence associated mRNAs, but upregulated activation, oxidative metabolism, protein synthesis and lineage associated transcripts. Although they showed no significant homing defects and had normal to elevated viability, Itpkb-/- HSC had a severely reduced competitive long-term repopulating potential. Aging Itpkb-/- mice lost hematopoietic stem and progenitor cells and died with severe anemia. Wildtype HSC normally repopulated Itpkb-/- hosts, indicating a HSC-intrinsic Itpkb requirement. In vitro, Itpkb-/- HSC had reduced cobblestone-area forming cell activity and showed increased stem cell factor activation of the phosphoinositide 3-kinase (PI3K) effector Akt. This was reversed by exogenous provision of the Itpkb product IP4, a known PI3K/Akt antagonist. Itpkb-/- HSC also showed transcriptome changes consistent with hyperactive Akt/mTOR signaling. Thus, we propose that Itpkb ensures HSC quiescence and function in part by limiting cytokine-induced PI3K signaling in HSC.
Blockade of PD-L1 accelerates the development of Sjögren’s syndrome in non-obese diabetic mice (HUM3P.256)
Authors: Zhou Jing , Jin Jun-O , Du Juan , Yu Qing
Published: January 2023

Programmed death-ligand 1 (PD-L1) is a B7-family member and plays a negative role in T cell -mediated immunity by binding to its receptors such as programmed death-1 (PD-1). Previous reports have shown enhanced expression of PD-L1 and PD-1 in the salivary gland of patients with Sjögren’s syndrome (SS), a prevalent systemic autoimmune disease characterized by progressive lymphocyte infiltration of exocrine glands and generation of autoantibodies, which together lead to impaired secretory function of salivary and lacrimal glands. However, the particular function of PD-L1 in SS disease development still remains poorly understood. Here we employed non-obese diabetic (NOD) mice, a model of SS-like disease to define the role of endogenous PD-L1 in the pathogenesis of SS. Administration of an anti-PD-L1 antibody to NOD mice enhanced leukocyte infiltration in salivary glands and elevated the levels of serum antinuclear antibodies. Importantly, PD-L1 blockade also has a crucial effect on the clinical phenotype of SS. We demonstrated blockade of PD-L1 markedly reduced salivary secretion, indicating an exacerbated secretory dysfunction. These results suggest that blockade of endogenous PD-L1 promotes the SS-characteristic pathological and clinical changes and accelerates SS development in NOD mice. Further understanding of the molecular mechanisms underlying the function of PD-L1 in SS could identify critical molecular targets for development of alternative therapeutic strategies.
Differentially expressed microRNAs decrease regulatory T cell development in multiple sclerosis (HUM3P.266)
Authors: Severin Mary , Guerau-De-Arellano Mireia , Lee Priscilla , Racke Michael , Lovett-Racke Amy
Published: January 2023

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the CNS. The disease is thought to be an autoimmune reaction mediated by autoreactive T cells. Interestingly, regulatory T cells (Tregs), protectors against autoimmune responses, are defective in MS patients. To better understand defects in the CD4 T cells of MS patients, a miRNA profiling study was performed on naïve CD4+ T cells of MS patients and healthy donors, identifying 85 differentially expressed miRNAs. By performing bioinformatics analyses, 23 miRNAs were predicted to target the TGFβ signaling pathway. TGFβ is critical for the development and function of Tregs. We hypothesized that dysregulated miRNAs in the naïve CD4+ T cells of MS patients target the TGFβ signaling pathway, resulting in defective Tregs and enhanced susceptibility to developing MS. To test this hypothesis, we conducted quantitative real-time PCR, flow cytometry, and luciferase assays to analyze TGFβ-associated genes and Treg markers in cells overexpressing TGFβ-targeting miRNAs. Our data indicate that 1) TGFβ signaling genes are decreased in MS patients’ naïve CD4 T cells, 2) several of the identified miRNAs directly target these TGFβ associated genes, and 3) overexpression of the TGFβ-targeting miRNAs cause a decrease in iTreg development in vitro and in vivo. Our data suggest that when overexpressed these identified miRNAs decrease TGFβ signaling and disrupt Treg development in multiple sclerosis.
Soluble mediators and clinical features predict transition to systemic lupus erythematosus classification in previously unaffected relatives of SLE patients (HUM3P.243)
Authors: Munroe Melissa , Young Kendra , Kamen Diane , Guthridge Joel , Niewold Timothy , Weisman Michael , Ishimori Mariko , Wallace Daniel , Gilkeson Gary , Olsen Nancy , Karp David , Harley John , Norris Jill , James Judith
Published: January 2023

Healthy relatives of lupus patients have an increased risk of developing SLE, but predictors and mechanisms of disease transition are unknown. Unaffected relatives of SLE patients (n=409) were enrolled in this follow-up study to identify factors associated with transition to SLE. Participants provided detailed demographic and clinical information, including the Connective Tissue Disease Screening Questionnaire (CSQ). Medical records were reviewed for ACR classification criteria. Plasma samples were tested for the presence of autoantibodies and soluble mediators. Forty-five relatives (11%) transitioned to classified SLE during follow-up (mean time = 6.4 years) who had more autoantibodies (p<0.0001) and higher pre-classification (baseline) CSQ scores (p<0.0001). Relatives who transitioned also had elevated baseline levels of inflammatory mediators, including BLyS, SCF, and IFN-associated chemokines (p<0.05), with concurrent decreased regulatory mediators, including TGF-β and IL-10 (p<0.05). Logistic regression analysis revealed that baseline ACR and CSQ scores (but not autoantibodies), and baseline plasma levels of SCF and TGF-β were significant and independent predictors of SLE transition. Thus, perturbations in immune-mediated processes precede SLE clinical classification and can help identify unaffected relatives at highest risk of future SLE classification for early intervention studies.
Autoantibody-positive healthy individuals display unique immune profiles that regulate autoimmunity (HUM3P.244)
Authors: Slight-Webb Samantha , Lu Rufei , Ritterhouse Lauren , Maecker Holden , Fathman C. , Merrill Joan , Guthridge Joel , James Judith
Published: January 2023

Antinuclear antibodies (ANA) are detected in up to 25% of the population, yet only 5-8% develop autoimmunity. Immunological differences between ANA+ healthy individuals and SLE patients may give us insight into mechanisms by which ANA+ individuals avoid autoimmunity or transition to SLE. European-American ANA+ individuals (n=31) were matched to ANA- controls and SLE patients. Serum cytokine profiles, lymphocyte subset frequency, and lymphocyte reactivity were analyzed. Numerous pro-inflammatory cytokines including IFNγ, IL-2, IL-5 and TNFα were elevated in the serum of ANA+ healthy individuals compared to ANA- controls; however, SLE patients had even higher cytokine levels compared to ANA+ healthy individuals (p>0.05). Interestingly, Type I IFNs, BLyS, IL-12p40 and SCF were only increased in SLE patients (p<0.05). Further, IL-1RA was decreased in both ANA+ and SLE individuals (p<0.0001). Total monocytes and memory B cells, were elevated in ANA+ individuals compared with ANA- controls while regulatory CD4+ T cells were decreased (p<0.05). Further, monocytes and B cells from ANA+ individuals had increased levels of pSTAT1 and pSTAT3 following IFN-α stimulation compared to controls (p<0.05). These data reveal ANA+ healthy individuals exhibit similar features to SLE patients with increased inflammation and loss of immune tolerance, but differ by the absence of elevated Type I IFNs, BLyS, IL-12p40 and SCF and severely decreased IL-1RA that contribute to disease pathogenesis.
CD3/SLAMF3-mediated co-stimulation promotes activation of the IL-2/IL-2R/STAT5 pathway and iTreg differentiation in naïve CD4+ T cells - implications for systemic lupus erythematosus (HUM3P.246)
Authors: Comte Denis , Kis-Toth Katalin , Karampetsou Maria , Yoshida Nobuya , Solomon Julie , Tsokos George
Published: January 2023

We examined SLAMF3-mediated co-stimulation on IL-2 response of CD4+ T cells from SLE patients. Stimulation of naïve CD4+ T cells with aCD3/aSLAMF3 upregulated surface CD25 (IL-2Ra) and CD122 (IL-2Rb), at higher level compared to aCD3/aCD28 activation. aCD3/aSLAMF3 co-stimulation of control naïve CD4+ T cells led to increased pSTAT5 levels compared to aCD3/aCD28 activation Although naïve CD4+ T cells from SLE patients displayed decreased pSTAT5 levels compared to control donors upon aCD3 or aCD3/aCD28 stimulation, co-stimulation of SLE T cells with aSLAMF3 restored pSTAT5 to normal levels. Total STAT5 levels remained unaltered after T cell activation. Exogenous IL-2 increased proliferation of aCD3/aSLAMF3-activated CD4+ T cells compared to aCD3/aCD28 stimulation. Naïve CD4+ T cells activated with aCD3/aSLAMF3 under iTreg polarizing condition expressed significantly higher levels of FoxP3, CD25 and CTLA-4 compared to aCD3/aCD28 activation. In conclusion, SLAMF3-mediated co-stimulation enhances CD4+ T cell response to IL-2 in SLE patients by acting on the IL-2/IL-2R/STAT5 signaling pathway, increases naïve CD4+ T cell proliferation in response to exogenous IL-2 and can promote iTreg phenotype generation. Of note, SLAMF3-mediated co-stimulation does not increase IL-2 by CD4+ T cells per se. Thus, aSLAMF3-based Ab therapy may emerge as a promising therapeutic agent in SLE by enhancing response to IL-2 and promoting iTreg differentiation.
ROCK2 signaling is required for development and function of T follicular helper cells (HUM3P.247)
Authors: Weiss Jonathan , Chen Wei , Nyuydzefe melanie , Flynn Ryan , Tonra James , Marusic Suzana , Blazar Bruce , Waksal Samuel , Zanin-Zhorov Alexandra
Published: January 2023

PD1+CXCR5+ T follicular helper (Tfh) cells are essential for generating high-affinity antibodies and B cell memory. But aberrant Tfh activity can drive autoimmune disease such as systemic lupus erythematosus (SLE) which is characterized by high levels of auto-antibodies and increased frequencies of IL-21+ T cells. Rho-associated kinase (ROCK) 2 was recently shown to be critical in regulating IL-21 secretion in both mice and humans. Here, we show that the PD1+CXCR5+ subset is a major source of IL-21 in human CD4+ T cells stimulated in vitro. By using KD025, a Kadmon orally available, selective ROCK2 inhibitor, we attenuated the development of PD1+CXCR5+ T cells by over 60% and the perpetuation of existing Tfh cells in a dose dependent manner. Targeting of ROCK2 concurrently regulates STAT3/STAT5 phosphorylation and transcription activity followed by a reduction of Bcl6 and up-regulation of Blimp1 protein levels in human T cells. Oral administration of KD025 reduced the percent of both Tfh and plasma B cells in spleens by more than 2 fold as well as germinal center size in the Mrl/lpr murine model of SLE. KD025 treated animals had a substantial improvement in both histological and clinical scores complimented by a reduction in pSTAT3 and BCL6 expression in addition to an increase in STAT5 phosphorylation. This data suggests that ROCK2 controls the function of Tfh cells and underscores the therapeutic potential of targeted ROCK2 inhibition in the clinic.
The impact of obesity on human γδ T cell function and homeostasis (HUM4P.271)
Authors: Dutt Shelley , Costanzo Anne , Han Peggy , Taylor Kristen , Fujioka Ken , Jameson Julie
Published: January 2023

CDC estimate the rate of obesity is on the rise, currently impacting one-third of the U.S. population. Obese patients exhibit side effects that include type 2 diabetes, chronic wounds and an increased vulnerability to infection. It is critical to understand how obesity impacts immune responses that affect these processes and create effective vaccines or antiviral treatments for obesity. We examined PBMC from donors with a lean BMI <25 or obese BMI >30. T cells were studied for responses to influenza virus A/PR/9/38 infection by monitoring upregulation activation markers and cytokine production, using flow cytometry. From these studies we have determined that obese donors have quantifiably fewer γδ T cells, which has implications for rapid immune responses to infection. γδ T cells are normally activated upon contact with infected influenza virus cells, however our findings show that IFN-γ production by γδ T cells is reduced in obese patients. In contrast there was no significant difference in the expression of cytotoxic markers such as granzyme B and NKG2D between lean and obese patients. Due to our finding, IL-2Ra is reduced on the cell surface and IL-2 is able to restore γδ T cell antiviral function,we hypothesize that γδ T cells in obese patients are lacking key T cell specific growth factor signals. Our findings show that gd homeostasis and antiviral function becomes impaired with obesity contributing to the increased susceptibility to infection found in obese subjects.
STAT5 tetramer formation is critical for mast cell function (HYP4P.312)
Authors: Kiwanuka Kasalina , Lin Jian-Xin , Leonard Warren , Ryan John
Published: January 2023

Signal transducers and activators of transcription (STATs) are latent transcription factors that are vital for cell survival, proliferation and function. Activated STAT proteins dimerize and translocate to the nucleus, where they bind to high-affinity motifs to modulate gene expression. These dimers can also form tetramers through an N-terminal domain-mediated oligomerization process, and bind to low-affinity tandem motifs. We previously generated Stat5a-Stat5b double knock-in (DKI) mice in which STAT5 proteins are phosphorylated and can form dimers but not tetramers. We have now found that bone marrow-derived mast cells from Stat5 DKI mice express FceRI and c-Kit at levels comparable to those from wild-type mice. However, these mast cells are defective in IgE-induced cytokine and chemokine production, and exhibit less SCF-mediated migration in vitro. Similarly, IgE-mediated passive systemic anaphylaxis is also altered in Stat5 DKI mice. Unraveling the role of STAT5 tetramers may reveal novel insights into mast cell function in allergic and inflammatory diseases.
The role of 1a,25-dihydroxyvitamin D3 in IgE-mediated mast cell activation (HYP4P.313)
Authors: Baker Bianca , Clark Nicholas , Boyan Barbara , Schwartz Zvi , Ryan John
Published: January 2023

As the prevalence of allergic diseases such as allergic asthma and rhinitis continues to increase, there is growing interest in novel approaches to blunt the inflammatory processes. IgE-mediated mast cell activation results in the release of histamine and other inflammatory mediators. Vitamin D has been linked to allergic disease, with vitamin D deficiency being linked to increased risk for development and severity of allergic disease. We assessed the effects of the Vitamin D metabolite 1a,25-dihydroxyvitamin D3 (1,25D3) on IgE-mediated mast cell activation in vitro. We find that 1,25D3 suppresses IgE-mediated inflammatory cytokine and chemokine production. Surprisingly, these effects do not require expression of the canonical receptor for 1,25D3 (VDR), as wild type and VDR KO mast cells are similarly suppressed by 1,25D3. Additionally, these effects appear to be somewhat stimulus-specific, as IL-33-mediated mast cell activation s not inhibited by 1,25D3. We postulate that the chaperone protein Pdia3, an alternative plasma membrane-associated receptor for 1,25D3, may be responsible for the VDR-independent, anti-inflammatory effects on IgE-mediated mast cell activation. Our data depict novel evidence that 1,25D3 can attenuate the pro-inflammatory mast cell response, and emphasize the possible utility of vitamin D supplementation for treatment of allergic and inflammatory diseases.
Augmentation of local granulopoiesis via TLR2 signaling to promote resolution of S. aureus-infected wounds (INC4P.322)
Authors: Falahee Patrick , Anderson Leif , Dahmubed Delsheen , Simon Scott
Published: January 2023

Staphylococcus aureus is the most frequent cause of skin and soft tissue infections seen in emergency departments across the United States and the increasing global prevalence of virulent antibiotic resistant strains such as Methicillin-resistant S. aureus (MRSA) represents a major public health concern. Polymorphonuclear leukocytes (PMN), or neutrophils, are first-line effector cells of the innate immune system that play a central role in S. aureus clearance. Defects in PMN number and function secondary to comorbidities such as diabetes or myeloablative chemotherapy frequently contribute to poor outcomes in patient populations. Using a transgenic mouse model of S. aureus-infected skin wounds, we recently reported on a novel innate immune mechanism whereby a subset of hematopoietic stem and progenitor cells (HSPC) traffic to the site of infection, undergo local granulopoiesis via a TLR2 and MyD88 signaling pathway, and play a critical role in the resolution of infection. Local produced PMN exhibit enhanced antibacterial function compared with those produced in bone marrow. We examined whether this pathway for extramedullary granulopoiesis is conserved in humans and found that treatment of bone marrow-derived CD34+ cells with the TLR2 agonist Pam3CSK4 drives efficient expansion of functional PMN in vitro. Studies are underway to improve the expansion efficiency of HSPC into PMN in order to study their impact on persistent and chronic MRSA infection in mouse wounds.
Manipulation of macrophage autophagy by Coxiella burnetii (INC4P.336)
Authors: Winchell Caylin , Graham Joseph , Voth Daniel
Published: January 2023

Q fever is an emerging zoonosis caused by the intracellular bacterium, Coxiella burnetii. Disease can present as an acute illness with debilitating flu-like symptoms or a chronic illness resulting in life-threatening endocarditis. Alveolar macrophages are the primary target cell for infection in which C. burnetii traffics through the phagolysosomal pathway. Upon fusion with lysosomes, C. burnetii uses a type IV secretion system (T4SS) to modify and expand this compartment into a replication niche termed the parasitophorous vacuole (PV). Autophagy is a eukaryotic process in which portions of bulk cytoplasm or specifically selected cargo are enclosed by autophagosomes and trafficked to lysosomes for degradation. Macrophages also use autophagy for clearance of bacteria and inflammasome regulation. We previously showed that recruitment of autophagosomes to the PV is dependent on T4SS activity. The cargo receptor p62 is also recruited to PV, therefore we investigated the role of p62 during infection. We found that p62 interacts with distinct host proteins during infection, including the cargo receptor NBR1. Additionally, we discovered a potential C. burnetii effector that may modulate interactions with autophagosomes. Finally, to assess the role of p62 in the innate immune response, we initiated studies to determine interactions with inflammasome components. These studies are defining a novel role for eukaryotic cargo receptors in intracellular bacterial pathogenesis.
A protective role for splenic neutrophils against Streptococcus pneumoniae infection (INC4P.338)
Authors: Deniset Justin , Kubes Paul
Published: January 2023

The spleen is integral for protection against infections to encapsulated bacteria, such as S.pneumoniae. A recently identified splenic resident neutrophil subset has been proposed to contribute to this protection by modulating the antibody production of marginal zone B cells. To date, little is known regarding the function and behavior of these neutrophils in vivo and whether they actively contribute to protection against infection. Employing intravital microscopy we aimed to characterize splenic neutrophil behavior and function. Under basal conditions, two subsets of neutrophils are observed, a circulating phenotype and a resident phenotype. Infection with S. pneumonia stimulates a robust recruitment of neutrophils to the spleen and an enhanced localization within the marginal zone. This recruitment is accompanied by neutrophil activation typified by a reduction in cellular movement. Neutrophil depletion within the spleen during infection resulted in a reduction in bacterial clearance and a significant decrease in survival. Depletion of marginal zone B cells mimicked these parameters, suggesting both cell types contribute to a common protective mechanism. Our findings suggest that splenic neutrophils play a role in the protection against S. pneumoniae infection. Future studies will determine the contribution of resident vs recruited neutrophils to this protection and the role of these populations in mediating S.pneumoniae specific antibody production.
Plasmacytoid dendritic cell-derived IFNα modulates Th17 differentiation during Bordetella pertussis infection in mice (INC4P.345)
Authors: Smith andrew , Wu Victoria , Segal-Abramson Tzvia
Published: January 2023

Whooping cough is a highly contagious respiratory disease caused by Bordetella pertussis (B. pertussis). Recent studies have revealed a central role for Th17 cells in the resolution of the infection. Emerging studies document that type I interferons (IFN) suppress Th17 differentiation and IL-17 responses in models of infection and chronic inflammation. Plasmacytoid dendritic cells (pDCs) are a major source of type I IFNs. Therefore, we hypothesize that during B. pertussis infection in mice, pDC derived IFNa inhibits a rapid increase in Th17 cells. We found that IFNa-secreting pDCs emerge in the lungs in the early stages of infection, while a robust rise of Th17 cells in the lungs is detected at 15 days post-infection (dpi) or later. The presence of IFNa led to ablation of Th17 differentiation and proliferation in vitro. Furthermore, blocking IFNa produced by pDCs, in vivo prior to B. pertussis infection resulted in increase of Th17 frequency, inflammation and reduced bacterial loads in the airways of infected mice. Altogether, this work provides evidence of an inhibitory role for pDCs and pDC-derived IFNa in modulating Th17 responses during the early stages of B. pertussis infection, which may explain the prolonged nature of whooping cough.
Chronic STING-dependent signaling in phagocytes aggravates TREX1-contingent inflammatory disease (INM2P.353)
Authors: Ahn Jeonghyun
Published: January 2023

Patients defective in the 3’ repair exonuclease 1 (Trex1) suffer from Aicardi-Goutieres Syndrome (AGS) characterized by lethal encephalitis caused by high levels of circulating type I IFN production. Trex1-/- mice similarly suffer a short life span due to inflammatory myocarditis. Our data indicates that Trex1 is responsible for eliminating DNA species that otherwise activate STING (Stimulator of Interferon Genes), a key innate immune regulator responsible for facilitating cytosolic DNA-mediated cytokine production. Loss of STING (Trex1-/- STING-/-) was observed to dramatically extend the lifespan of Trex1-/- mice due to the prevention pro-inflammatory gene induction. However, we noted that Trex1-/- macrophages did not exhibit an augmented ability to produce cytokines following exposure to STING-dependent activators, but rather appeared chronically activated, exhibiting high basal level cytokine production. The source of STING activator was found to involve intrinsic genomic DNA left over from hematopoetic cell division processes that leaked into the cytoplasm, the elimination of which usually require Trex1. Accordingly, Trex1-manifested disease could be alleviated following adoptive transfer of normal bone marrow into Trex1-/- mice. Collectively, our data indicates that STING is responsible for pro-inflammatory gene induction induced by intrinsic self-DNA in Trex1 deficient mice and provides new insight into the molecular mechanisms underlining self-DNA-mediated disease.
Blockade of either SYK or BTK signaling pathways inhibits microparticle-induced type 2 inflammatory response (IRM11P.628)
Authors: Mishra Pankaj , Palma Mark , Millman Ariel , Hallab Nadim , Beebe Kathleen , Benevenia Joseph , Gause William
Published: January 2023

Wear debris microparticle release associated with total joint arthroplasty may contribute to harmful inflammation, osteolysis and implant failure. We examined the role of different molecular signaling pathways in microparticle-mediated inflammation. Intraperitoneal inoculation of cobalt chrome (CoCr) in mice induced a robust innate type 2 response with increases in Th2 cytokines, M2 macrophages, eosinophils and neutrophils compared to controls given vehicle only. Furthermore, this type 2 innate particle-induced response was independent of MYD88/TRIF and partially dependent on Caspase-1 signaling pathways. Blockade of the PGE-2 signaling pathway by administration of Aspirin or Celecoxib prior to CoCr inoculation caused a significant reduction in both neutrophils and macrophages with no effect on eosinophils and Th2 cytokines compared to the mice treated with CoCr + vehicle. Blockade of the SYK signaling pathway with the inhibitor BAY61-3606 or the BTK signaling pathway with Ibrutinib completely abrogated the CoCr-mediated innate inflammation and Th2 cytokines. These studies indicate that the type 2 immune response caused by solid particles is partially modulated by Caspase-1 and PGE-2 pathways and largely abrogated by blockade of upstream SYK and BTK signaling pathways. These studies provide potential targets for controlling the wear debris-mediated inflammation in synovial tissues that can contribute to aseptic loosening and implant failure.
C/EBPβ controls mucosal fungal immunity through regulation of β-defensins (IRM11P.630)
Authors: Simpson-Abelson Michelle , Childs Erin , Bishu Shrinivas , Solis Norma , Uematsu Satoshi , Conti Heather , Akira Shizuo , Filler Scott , Gaffen Sarah
Published: January 2023

Th17 cells and IL-17 play essential host-defensive roles in immunity to pathogens, particularly the commensal microbe Candida albicans. Although best known for activation of NF-κB, IL-17 potently activates CCAAT enhancer binding protein transcription factors, which are required for induction of many IL-17 target genes. C/EBPβ is translated into 3 distinct protein isoforms whose expression is regulated by the differential use of different translational initiation start sites, known as LAP* (38kDa), LAP (35kDa) and LIP (22kDa). We have shown that IL-17 induces the alternative translation of C/EBPβ, inducing a marked increase in LAP* and LIP. However, the functional significance of differential C/EBPβ isoform expression remains enigmatic. To determine the biological significance of C/EBPβ alternative translation, we used C/EBPβ-/- mice and a knockin that cannot generate the LAP isoform (C/EBPβM20A). We then assessed susceptibility of these mice to oral C. albicans infection. We found that C/EBPβ-/- but not C/EBPβM20 mice were highly susceptible to disease indicating that LAP is dispensable but C/EBPβ is required. In evaluating a panel of downstream genes to elucidate mechanisms of C/EBPβ-dependent immunity, the gene encoding β-defensin 3 (BD3) was the only IL-17 target gene strongly downregulated in susceptible C/EBPβ-/- mice. These results highlight a key role for C/EBP in antifungal immunity mediated by IL-17 and a previously unrecognized role for C/EBPβ in BD3 expression.
Immune modulatory mechanisms of lanthionine synthetase C-like protein 2 (IRM12P.656)
Authors: Washington Philipson Casandra , Heltzel Chase , Eden Kristin , Hontecillas Raquel , Bassaganya-Riera Josep
Published: January 2023

Lanthionine synthetase C-like protein 2 (LANCL2) has emerged as a novel therapeutic target for metabolic, infectious and immune-mediated diseases. We previously demonstrated that directly targeting LANCL2 through treatment with abscisic acid or 61610 results in efficacious anti-inflammatory responses during DSS-induced colitis however molecular mechanisms underlying this immunomodulatory function remain elusive. Here we report for the first time the use of LANCL2-deficient mice to investigate the mechanism of action and cell specificity of the protein. At the cellular level, bone marrow derived macrophages (BMDM), RAW cells, and gastric epithelial cells express comparable levels of basal LANCL2 mRNA. LPS-mediated inflammation in BMDM increased both mRNA and protein levels of LANCL2 that peaked 6 hours post-challenge and subsequently returned to normal levels after 12 hours. Similarly, DSS-induced colitis induced expression of LANCL2 in the colons of wild-type (WT) mice and correlated with elevated levels of proinflammatory cytokines. Interestingly, the absence of LANCL2 did not cause significant alterations in infiltrating cellular phenotypes however LANCL2-deficiency permitted exaggerated IL17 and IFNγ expression in colonic tissue when compared to WT counterparts at day 7 post-challenge. In conclusion, our findings indicate that LANCL2 is a novel regulator of inflammation that functions predominantly at the molecular level to down-regulate excessive inflammatory responses.
Elucidating a role for a hematopoietic-tropic microRNA in T cell (IRM12P.650)
Authors: Menoret Antoine , Kumar Sanjeev , Palakodeti Dasaradhi , Graveley Brenton , Vella Anthony
Published: January 2023

To gain new insight into how miRNA impact T cell responses, mice were immunized with costimulation and adjuvant to generate potent T effector responses. We generated a cDNA library from lymphoid tissue. Using sequencing of size-fractionated cDNA we cloned 6 potential new miRNAs with characteristic stem loop folding. One of these candidates, termed miR-R89 but not yet registered in miRBase, was unique since its expression was hematopoietic specific and regulated during immune activation. The targets and function of miR-R89 are currently unknown, but our preliminary data show it is expressed in a T cell clone known to cause experimental autoimmune encephalitis. Transfection with a locked nucleic acid (LNA) mimic of miR-R89 in EL4 thymoma cells increased IL-2 secretion after PMA-ionomycin (PI) stimulation compared to transfection with control LNA. RNAseq analysis of the transfected cells revealed that mRNA expression of two transcription factors involved in metabolism, Tfe3 and Tef, was reduced by miR-R89 mimic. Interestingly, 3’UTR mRNA of Tfe3 and Tef have several theoretical binding sites for miR-R89. Treatment of PI-stimulated EL4 cells with UK5099, an inhibitor of mitochondrial pyruvate transport and metabolism, mediated a similar increase of IL-2 secretion than observed after transfection with miR-R89. These data suggest the putative miRNA miR-R89 could target Tfe3 and Tef mRNA and function as a metabolic regulator of T cell activation.
The level of antigen expression in the liver predicts the fate and functional outcome of antigen-specific CD8 T cells (LYM5P.706)
Authors: Bertolino Patrick , Wong Yik Chun , Tay Szun Szun , McDonald David , Sierro Frederic , Roediger Ben , Wood Nicole , McGuffog Claire , Bremner Kate , Alexander Ian , Weninger Wolfgang , McCaughan Geoffrey , Bowen David
Published: January 2023

A wide spectrum of CD8 T cell responses can be elicited against antigens (Ag) expressed in the liver, ranging from tolerance to full effector function. Identifying the factors involved would allow us to predict the outcome of liver transplantation and viral hepatitis. To investigate how CD8 T cells respond to liver Ags, we have developed recombinant adeno-associated viral vectors that, when injected into mice, allow de novo Ag expression in a variable number of hepatocytes. Within the first day of Ag expression by at least 70% of hepatocytes, most naïve Ag-specific CD8 T cells were activated in the liver via direct presentation by hepatocytes. CD8 T cell activation in lymph nodes and spleen via cross presentation was detected a day later, suggesting that primary activation of CD8 T cells against hepatocyte-expressed Ag is compartmentalised. These T cells proliferated, developed into CTLs in the first week but were unable to kill all Ag-expressing hepatocytes. Most effector T cells were rapidly deleted while those surviving became exhausted and unresponsive over time. In contrast, when less than 10% of hepatocytes expressed the targeted Ag, CD8 T cell were gradually activated in liver and lymphoid tissues but were not deleted. Instead, they eliminated all Ag-expressing hepatocytes and maintained their effector function overtime. In summary, this study shows that intrahepatic Ag load dictates both the fate and function of CD8 T cells recognising hepatocyte-expressed Ags.
Resveratrol regulates the miR-124/sphingosine kinase 1 axis in encephalitogenic T cells and protects from experimental multiple sclerosis (LYM5P.709)
Authors: Gandy K. , Nagarkatti Mitzi , Nagarkatti Prakash
Published: January 2023

Resveratrol (RES) is a naturally-occurring phytoestrogen with anti-inflammatory properties. In this study, we used a mouse model of multiple sclerosis, experimental autoimmune encephalitis (EAE), to evaluate the effect of RES on T helper cell function. We hypothesized that RES protects against EAE via regulation of encephalitogenic T cell miRNA expression. We found that RES treatment diminished clinical parameters of EAE and altered immune cell composition and activation in the brain. Analysis of brain-derived CD4+ T cells identified significant up-regulation of several miRNAs with RES treatment. In silico analysis of target genes revealed overlap with cell growth and proliferative functions. We identified brain-specific alteration of the miR-124/sphingosine kinsase 1 (SK1) axis in RES-treated mice, with no significant changes in peripheral CD4+ cells. In vitro RES treatment of peripherally-derived mononuclear cells had no effect on miR-124 expression, supporting the brain-specific regulation of CD4+ T cell miRNA expression by RES. Lastly, we evaluated the consequence(s) of RES-mediated alteration of the miR-124/SK1 axis by assessing encephalitogenic CD4+ T cell function. The current study demonstrates a role for the miR-124/SK1 axis in RES-mediated protection from EAE, suggesting that RES may be an ideal candidate for the treatment of neuroinflammatory diseases.
Calmodulin and PI(3,4,5)P3 cooperatively bind to the Itk Pleckstrin homology domain to promote efficient calcium signaling and IL-17A production in T cells (LYM5P.710)
Authors: Huang Yina , Wang Xinxin , Boyken Scott , Shea Madeline , Andreotti Amy
Published: January 2023

Precise regulation of the kinetics and magnitude of Ca2+ signaling enables this signal to mediate diverse responses, such as cell migration, differentiation, vesicular trafficking, and cell death. Here, we showed that the Ca2+-binding protein calmodulin (CaM) acted in a positive feedback loop to potentiate Ca2+ signaling downstream of the Tec kinase family member Itk. Using NMR (nuclear magnetic resonance), we mapped CaM binding to two loops adjacent to the lipid-binding pocket within the Itk pleckstrin homology (PH) domain. The Itk PH domain bound synergistically to Ca2+/CaM and the lipid phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3], such that binding to Ca2+/CaM enhanced the binding to PI(3,4,5)P3 and vice versa. Disruption of CaM binding attenuated Itk recruitment to the membrane and diminished release of Ca2+ from the endoplasmic reticulum. Moreover, disruption of this feedback loop abrogated Itk-dependent production of the proinflammatory cytokine IL-17A (interleukin-17A) by CD4+ T cells. Additionally, we found that CaM associated with PH domains from other proteins, indicating that CaM may regulate other PH domain-containing proteins.
Determining the contribution of antigen-specific, CD4+ recent thymic emigrants to Salmonella persistence in vivo (LYM5P.661)
Authors: Goggins J. , Kurtz Jonathan , Israel Kate , McLachlan James
Published: January 2023

Typhoid fever, caused by infection with Salmonella bacteria affects approximately 21 million people annually, killing nearly 200,000. Recent thymic emigrants (RTEs) are the youngest subset of peripheral T cells, which are known to differ in how they functionally contribute to the naïve T cell pool. This distinctive cell population is known to contribute to the maintenance of T cell receptor diversity in the peripheral T cell pool, but their involvement in combating chronic bacterial infections, such as typhoid fever, has not been explored. In the present study, we hypothesized that RTEs are essential contributors to the CD4 T cell response during persistent Salmonella Typhimurium (ST) infection, which predominantly relies on helper T cell immunity to control bacteria. Here, we show that thymectomy of adult mice prior to oral infection with ST led to significantly increased bacterial burdens in the spleen, mesenteric lymph nodes and liver 30 days post-infection compared to sham surgical controls, suggesting Salmonella-specific RTEs may contribute to bacterial clearance. Surprisingly, existing peripheral Salmonella-specific T cells also increased significantly in number, implying that these cells are somehow incapable of controlling infection in the absence of RTEs. The results of this study provide insight into how new pathogen-specific T cells leaving the thymus participate in combatting a persistent bacterial infection.
Effects of natural supplementation on human T cell longevity pathways (LYM6P.723)
Authors: Valenzuela Hector , Casarez Nicholas , Hom Lisa , Daoudi Sarah
Published: January 2023

Nutraceuticals are natural compounds that contain both nutritional as well as medicinal benefits. Various nutraceuticals (curcumin and resveratrol) obtained from plant extracts have been shown to improve fitness and extend lifespan by various mechanisms. In addition other compounds have also been shown to extend lifespan (rapamycin, spermidine and nicotinamide mononucleotide). Although the end result for these compounds maybe extension of lifespan, comparison of their signaling pathways remains to be determined. The objective of our research was to compare the signaling pathway of these compounds on both normal human T cells and Jurkat cell lines. We used RT-PCR to measure gene expression of NF-Κβ, AMPK, sirtuin 1-7 and autophagy specific genes (ATG5 and MAPILC3B/LC3) for all of these compounds. In addition, we show a direct correlation between concentrations of treatment and the upregulation of gene expression to the viability of the cells.