bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolic changes induced by T cell-mediated cutaneous vaccinia virus clearance
Authors: Pei Luxin , Jarmusch Alan K , Hickman Heather D
Published: August 2023

Viral infections induce prominent alterations to the host metabolism. Vaccinia virus (VACV) has been used as a live vaccine preventing smallpox and monkeypox. However, much remains unknown about the metabolic regulations of the adaptive immune response mounted to VACV infection in the skin.

To survey the metabolic changes during VACV clearance, we performed a longitudinal metabolomics study based on our cutaneous VACV infection murine model. We adoptively transferred OT-I CD8+ T cells into Rag−/− mice, then infected one ear with VACV expressing SIINFEKL and VACV lacking SIINFEKL in the contralateral ear. Ears were harvested on 0,5,6,7,8,10 days post infection (dpi), which were homogenized and methanol extracted prior to untargeted mass spectrometry analyses.

Metabolomics profiling revealed that VACV infection induced significant metabolic changes over time in the ear tissue. Interestingly, the metabolic profile after viral clearance was still distinct from uninfected control group. The levels of phenylalanine (adj. p=0.032), oleic acid (adj. p=0.011), S-lactoylglutathione (adj. p=0.022), and hypoxanthine (adj. p=0.039) were significantly altered between samples undergoing T cells-mediated killing and those without. Furthermore, glutamic acid level was significantly different (adj. p=0.036) between groups with and without T cells, suggesting T cell utilization during VACV clearance.

Overall, our results indicated that VACV infection caused significant and persistent metabolic perturbation in the skin tissue environment. Antiviral T cells that mediate VACV clearance may require specific metabolites or metabolic pathways to regulate cytotoxic functions.
Quantitation of Immune Synapse Duration Supports the Contribution of Perforin Haploinsufficiency to Hyperinflammation
Authors: Frank-Kamenetskii Anastasia M , Klinghoffer Hannah , Dang Vinh , Varghese Jemy , Canna Scott
Published: August 2023

Hyperinflammation is an acute state that occurs in a variety of infectious, inflammatory, and genetic contexts. Familial Hemophagocytic Lymphohistiocytosis (FHL) is a rare Inborn Error of Immunity resulting from biallelic disruption of perforin or other genes necessary for granule-mediated cytotoxicity. Some reports suggest that heterozygous defects in FHL genes contribute to hyperinflammation in concert with infection or rheumatic diseases. Most known causes of hyperinflammation arise from abnormal regulation of the Immune Synapse (IS) between cytotoxic CD8 T-cells and target cells. Using live imaging, we sought to assess the effects of Perforin and other putative contributors to hyperinflammation on IS duration and magnitude.

We evaluated the IS between CD8 T cells from mice with different T-cell receptor signal strengths (OT1 vs P14) and various target cell types, as well as that between Prf1+/+, +/−, and −/− P14 cells and BMDCs. IS duration was calculated based on time between killer cell calcium flux and target cell Propidium Iodide uptake, and cytokine ELISA of supernatants was performed.

We found that OT1, but not P14, CD8 T-cells could kill MC57 fibrosarcoma cells, but both were able to kill BMDCs. Moreover, both complete Prf1 deficiency and Prf1 haploinsufficiency prolonged the IS and increased cytokine production. Excess IL18, a hyperinflammatory susceptibility factor that synergizes with Prf1 deficiency in vivo to drive spontaneous hyperinflammation, shortened IS duration but dramatically increased IFN-γ production. Thus, showing that our in vitro IS quantitation model is sensitive to small changes that may be important contributors to pathogenic T-cell activation and life-threating hyperinflammation.

Supported by grants from NIH (R01 HD098428-03-04), Children’s Hospital of Philadelphia (IDF FY21-FY24), and from Histiocytosis Association (FP00039723_A1)
A commensal-derived exopolysaccharide regulates ILC3-mediated diseases
Authors: Tan Chin Yee , Theriot Barbara S , Rao Meghana V , Surana Neeraj K
Published: August 2023

The gut microbiota plays a fundamental role in controlling many facets of human physiology, with impacts on the immune system being chief among them. Although there have been increasing successes in identifying disease-regulating commensal bacteria, the mechanisms by which they work remain poorly understood. We previously identified Clostridium immunis, a new human-derived commensal bacterial species that modulates colitis. Here we identify the immunological factors and specific bacterial determinant required for its disease modulating activity. We find that C. immunis directly regulates the effector function of group 3 innate lymphoid cells (ILC3s), with altered levels of IL-22 and GM-CSF in states of health and inflammation, respectively. This impact on ILC3s serves as a common immunological node for modulation of visceral adiposity and intestinal inflammation. Surprisingly, the two bacterial species most closely related to C. immunis lack this ILC3-modulating activity. Guided by comparative genomics, we purified an exopolysaccharide (EPS) that recapitulates the immunomodulatory activity of C. immunis in vitro and in vivo. Structural analysis revealed that phosphocholine on the EPS correlated with activity, and deletion of the C. immunis phosphocholine biosynthesis locus (LicABC) resulted in functionally inactive EPS. Our results reveal that a phosphocholine moiety present on a bacterial polysaccharide is critically required to orchestrate ILC3 effector function and regulate intestinal homeostasis. Considered together, we provide genetic and structural insight into how a human commensal bacterium provides a unique immunomodulatory function.

Supported by a predoctoral fellowship from the American Heart Association (897275), a postgraduate scholarship from the Tan Kah Kee Foundation, and a Development Grant from the National University of Singapore (to C.Y.T) Supported by grants from NIH (NIH R03 AI142341), The Hartwell Foundation, and as a Translating Duke Health Scholar and as a Whitehead Scholar (to N.K.S.).
Gut microbiome-derived isodeoxycholic acid promotes mucosal immune non-responsiveness
Authors: Ionescu Edward , Arnold Jack , Mimee Mark , Nagler Cathryn R
Published: August 2023

Perturbations to the commensal gut microbiota are associated with the increasing prevalence of food allergies. Our lab has reported that colonization of germ-free mice with feces from healthy, but not cow’s milk allergic, infants protects against anaphylaxis to a cow’s milk allergen. Protection was associated with higher levels of the secondary bile acid (SBA) isodeoxycholic acid (isoDCA) in the feces of healthy-colonized mice. IsoDCA has been reported to induce regulatory T cells (Tregs) which suppress inflammation. We hypothesize that isoDCA-driven induction of mucosal Tregs promotes non-responsiveness and protects against food allergy. To study this, we identified two Clostridia species, Peptacetobacter hiranonis and Ruminococcus gnavus, that together produce isoDCA from taurocholic acid (TCA), an abundant host bile acid. Using a Group II intron-based system, we engineered R. gnavus Δrumgna_00694, a functional knock-out of a 3β-hydroxysteroid dehydrogenase required to produce isoDCA. In our system, P. hiranonis converts TCA to deoxycholic acid (DCA) and R. gnavus WT, but not Δrumgna_00694, converts DCA to isoDCA. Ongoing experiments will compare isoDCA-dependent Treg induction in gut-associated lymphoid tissues of mice co-colonized with P. hiranonis and R. gnavus WT or Δrumgna_00694. Once optimized, this system can be used in our allergic sensitization model to investigate the role of SBA in protection against food allergy and maintenance of intestinal homeostasis.
Commensal-specific CD4 T cells can promote inflammation in the central nervous system via molecular mimicry
Authors: Sano Teruyuki , White Zachary , Cabrera Ivan
Published: August 2023

Commensal bacteria are critical regulators of both tissue homeostasis and the development and exacerbation of autoimmunity. However, it remains unclear how the intestinal microbiota contributes to inflammation in tissues such as the central nervous system (CNS) where these microbes are typically absent and whether T cell receptor (TCR) specificity for commensal-derived antigens is important to the development of tissue inflammation-related outcomes. Here, we found that ileum- and cecum-colonizing segmented filamentous bacteria (SFB)-specific T cells (clone TCR7B8) can infiltrate the CNS wherein they can be reactivated and produce high levels of inflammatory cytokines including IFNγ, IL-17A, TNFα, and GM-CSF in the absence of regulatory T cells. In contrast, other SFB-specific T cells (clone TCR1A2) recognizing an epitope in which 8/9 amino acids overlap with those recognized by TCR7B8 failed to induce such neuroinflammation. Despite their similar SFB-derived peptide antigen targets, TCR7B8 was found to recognize peptides derived from host proteins including receptor tyrosine-protein kinase ErbB2, trophinin 1, and anaphase-promoting complex subunit 2 in vitro, whereas TCR1A2 did not, indicating that TCR7B8 induces CNS inflammation via molecular mimicry. Immune checkpoint blockade accelerated TCR7B8-mediated CNS inflammation. Together, our findings reveal a potential mechanism whereby gut commensal-specific T cells are dysregulated, infiltrate into the CNS, and contribute to extraintestinal inflammation.

Supported by grants from UIC start-up funds and UIC COVID Relief Funding.
Intake of low dietary fiber changes microbiota composition and reduces IL-10 production from small intestinal T cells
Authors: Rivera-Rodriguez Dormarie E , Rodriguez-Marino Naomi C , Royer Charlotte J , Yaceczko Madelyn , Cervantes-Barragan Luisa C R
Published: August 2023

Regulatory immune responses are crucial for intestinal homeostasis against stimuli like diet and microbiota. The microbiota influences the regulatory immune response by inducing colonic Foxp3+ regulatory T cells and other lymphocytes by metabolizing diet-derived products. For example, fiber-fermenting microbiota process fermentable fiber into metabolites like short-chain-fatty-acids (SCFA) and help activate IL-10-producing T cells. However, studies showed that high-fat (low-fiber) diet alters the abundance of Treg-inducing commensal bacteria promoting intestinal inflammation. Previous studies in our lab showed that low fermentable fiber affects the generation of many intestinal T cells, especially intraepithelial CD4+CD8aa+ T cells (DP IELs) and CD8aa IELs. However, it is still uncertain how dietary fiber affects small intestinal T cell function, including IL-10 production. To address this, we fed IL-10-GFP reporter mice with a regular chow diet or an isocaloric isonitrogenous diet that replaced fiber with non-fermentable fiber (LFF). After four weeks, the lymphocytes and microbiota from the small intestine were analyzed. Our preliminary findings show that IL-10 mRNA expression and IL-10-GFP production from intestinal lymphocytes was lower in mice fed LFF diet when compared to the control. Moreover, 16S RNA sequencing of intestinal contents showed changes in relative frequencies of several OTUs between diets. These results suggests that certain bacterial species affected by the diet could be significant in promoting IL-10 production by small intestinal T cells. Further studies will seek to understand how fiber plays a role in orchestrating the host-microbiota interactions to maintain intestinal tolerance.

These studies were conducted at Emory University and funded by the following NIH grants: 1R01DK129950-01 (NIDDK-NIH from LCB); 5T32AI106699-08 (NIDDK-NIH from Antimicrobial Resistance and Therapeutic Discovery Training Program)
Diversity of gut commensal flagellins results in distinct intestinal epithelial cell immune responses
Authors: Jennings Melissa S , Duck Wayne , Zhao Qing , Alexander Katie , Muir Rachel Q , Hsu Jung-Shan , Klocke Barbara J , Lee Goo , Elson Charles O , Maynard Craig L
Published: August 2023

Flagellins from bacteria in the Lachnospiraceae family are immunodominant antigens in inflammatory bowel disease. Flagellin is the basic unit of flagella which is used for bacterial motility and is recognized by toll-like receptor 5 (TLR5). Due to the diversity in flagellated organisms that reside in proximity to the intestinal epithelium, we asked whether this resulted in distinct interactions between commensal flagellins and the immune system. Analysis of over 200 flagellins, primarily from Lachnospiraceae organisms, revealed a divergence in peptide sequences in specific non-conserved regions of the D0 domain that broadly separate the flagellins examined into 2 clusters which we refer to as Group 1 and Group 2 flagellins. We stimulated HEK-Blue mTLR5 cells and murine intestinal epithelial cells (IECs) with selected Group 1 or Group 2 flagellins to assess TLR5 activity and IEC production of lipocalin-2 (Lcn-2) - a biomarker of inflammation - respectively. Group 2 flagellin strongly activated TLR5 signaling in the mTLR5 HEK cell line, even more so than Salmonella typhimurium (S. typhi) flagellin. Conversely, cells stimulated with the Group 1 flagellin exhibited significantly reduced TLR5 signaling relative to both Group 2 and S. typhi flagellins. While IEC stimulation with Group 1 flagellin resulted in undetectable Lcn-2, stimulation with Group 2 flagellin led to robust Lcn-2 production; more so than stimulation with S. typhi flagellin. These findings demonstrate that flagellins of commensal bacteria elicit differential IEC response via selective activation of TLR5. These functional differences are dependent on specific motifs in non-conserved regions of their D0 domains.

Supported by R01 AI162736, R21 DK118386, T32 AI007051.
Commensal bacteria regulate host TGF-β and retinoic acid metabolism to promote intestinal homeostasis
Authors: Puente Armando Andres , Nagler Cathryn
Published: August 2023

TGF-β signaling and retinoic acid (RA) metabolism are essential to intestinal homeostasis, but the mechanisms by which the microbiota influence these host processes are not clear. We have previously reported a mouse model of cow’s milk allergy (CMA) in which germ-free mice were colonized with the fecal microbiota of healthy infants or infants with CMA. That study revealed that Tgfbr3 was part of an ileal transcriptomic signature that distinguished healthy from CMA-colonized mice. Follow-up experiments using RT-qPCR confirmed that Tgfbr3 expression was significantly higher in the ileum of healthy-colonized than CMA-colonized mice. Tgfbr3 encodes a TGF-β co-receptor, and its expression is reported to be induced by RA; genes in the CMA transcriptomic signature encode enzymes with RA catabolic activity (Cyp2c29, Cyp2b10, Cyp3a59, and Akr1c19). The expression of RA-catabolic enzymes and concurrent downregulation of Tgfbr3 led us to hypothesize that these distinct microbiotas differentially modulate host RA metabolism, and consequently influence TGF-β signaling through the induction of Tgfbr3. In support of this hypothesis, we found lower ileal expression of Aldh1a1 in CMA-colonized mice, suggesting a lower capacity to produce RA. Furthermore, Tgfbr3 expression correlated with the transcript levels of TGF-β pathway mediators, Smad3 and Smad7, both of which are increased in the ileum of healthy-colonized mice. Ongoing experiments will use single cell RNA-sequencing to identify the cell populations responsible for the differential expression of Tgfbr3 in the ileum and to characterize the microbial mechanisms responsible for modulating host RA metabolism.

Supported by NIH R01 AI146099

Supported by NIH R01 AI146099
Microbiota engage a HDAC3 rheostat to shape ILC3 metabolism and function in the gut
Authors: Jensen Samantha N , Teng Fei , Alenghat Theresa , Lazar Mitchell A , Sonnenberg Gregory F
Published: August 2023

Group 3 innate lymphoid cells (ILC3s) respond to microbial colonization of the intestine to coordinate tissue health. This process involves interpretation of host- or microbial-derived signals that must be tightly regulated to preserve immunologic homeostasis. Here we examine a key epigenomic regulator that is responsive to microbial-derived factors, histone deacetylase 3 (HDAC3), and find that lineage-specific deletion results in reduced ILC3s and increased susceptibility to intestinal damage. In contrast, the lineage-specific deletion of an essential corepressor, NCOR1, results in elevated ILC3s and enhanced protection from intestinal damage. This indicates that HDAC3 mediates both a supportive deacetylase-independent role and a repressive deacetylase-dependent role in ILC3s. To explore genes promoted by HDAC3, we performed RNA sequencing and found that HDAC3 deficient ILC3s exhibit impaired transcription of genes involved in polyamine synthesis including the polyamine rate limiting enzyme ornithine decarboxylase 1 (ODC1) and spermine oxidase (SMOX). At steady state, ILC3s uniquely express high levels of ODC1 and SMOX compared to other lymphocytes, and transcription of these genes is influenced by IL-23 and the presence of the microbiota. Overall, this indicates that HDAC3 is a rheostat of ILC3 functionality in the intestine, with the hypothesis that HDAC3/NCOR1 limit tissue protective ILC3 responses, and NCOR1 independent HDAC3-mediated transcription of polyamine synthesis genes promotes ILC3 tissue protective responses.

Supported by NIDDK National Research Service Award (NRSA) T32 Diversity Administrative Supplement (NOT-DK-21-016), R01AI143842, R01AI123368, R01AI145989, U01AI095608, R21CA249274, R01AI162936, R01CA274534 and the NIAID Mucosal Immunology Studies Team (MIST), an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund, Linda and Glenn Greenberg, the Dalton Family Foundation, and the Roberts Institute for Research in IBD. G.F.S. is a CRI Lloyd J. Old STAR.
Group-3 innate lymphoid cells protect from intestinal inflammation via syndecan-4
Authors: Fiedler Brooke Elizabeth , Zhou Wenqing , Joseph Ann , Sonnenberg Gregory F
Published: August 2023

Group-3 innate lymphoid cells (ILC3s) are a recently described set of tissue resident immune cells which are enriched in the intestine, express RORγt, and secrete a variety of effector cytokines to orchestrate homeostasis, inflammation, and immunity. Crucially, intestinal ILC3 function and frequency is dysregulated during inflammatory bowel disease (IBD). However, it remains unclear how ILC3s sense and respond to the heterogeneity of signals present in the intestinal microenvironment or how this cell type becomes disrupted during intestinal inflammation.

Recently, a single-cell RNA-sequencing analysis in our laboratory identified that syndecan-4 is highly expressed within ILC3s. Syndecans are a class of heparan sulfate proteoglycans which can become dysregulated in the intestinal epithelium and the lamina propria of IBD patients. Further, mice lacking global expression of syndecan-4 show a heightened susceptibility to models of intestinal inflammation. However, cell-specific and molecular mechanisms for this protection have yet to be elucidated. Here, we unexpectedly identify that syndecan-4 is significantly enriched among ILC3s relative to other intestinal immune cells. IL-1 selectively upregulates ILC3-specific syndecan-4, but during acute and chronic mouse modeling of intestinal inflammation, ILC3 expression of syndecan-4 is reduced. Mice lacking ILC3-specific syndecan-4 exhibit exacerbated susceptibility to intestinal inflammation resulting in greater loss of intestinal epithelial architecture compared to controls. Collectively, our data indicate that syndecan-4 represents a novel pathway by which ILC3s orchestrate intestinal health and limit tissue damage during inflammation.

Research in the Sonnenberg Laboratory is supported by the National Institutes of Health (R01AI143842, R01AI123368, R01AI145989, U01AI095608, R21CA249274, R01AI162936 and R01CA274534), the NIAID Mucosal Immunology Studies Team (MIST), an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund, Linda and Glenn Greenberg, the Dalton Family Foundation, and the Roberts Institute for Research in IBD. B.F. is supported by the NIAID of the National Institutes of Health training grant T32 (5 T32 AI 134632-5). G.F.S. is a CRI Lloyd J. Old STAR.
Lactobacillus johnsonii supplementation modulates functional response of bone-marrow derived dendritic cells in respiratory allergy and viral infection
Authors: Martinez Llilian Arzola , Ethridge Alexander D , Rasky Andrew J , Morris Susan , Fonseca Wendy , Lukacs Nicholas W
Published: August 2023

The immunomodulatory properties of probiotics are particularly important at the establishment of Th1-Th2 immune balance early in life. A skewing to Th2 immune response driven by dendritic cell regulation can lead to airway alteration during respiratory allergy and respiratory virus infection. Previous work from our lab have determined that oral supplementation of mice with Lactobacillus johnsonii (Lj) resulted to be protective against viral and allergen airway exposure. In order to further analyze whether Lj supplementation of moms could alter offspring responses to allergic disease, female mice were supplemented daily with Lj for a week before mating. Allergen-sensitized offspring from Lj supplemented-mothers were protected from airway hyperreactivity, had a diminished mucus production and shown a reduced allergen-restimulated Th2 cytokine response upon later life allergen-induced responses. Maternal Lj supplementation changed the functional response of bone-marrow derived dendritic cells (BMDC) activated in vitro with respiratory syncytial virus (RSV), decreased expression of pathological cytokines and chemokines in non-sensitized pups, but increased protective innate cytokines expression in allergic offspring. Neonates supplemented directly with Lj before early life RSV infection altered long term inflammatory activity of BMDC by reducing expression of proinflammatory innate cytokines. These results have shown the immunomodulatory potential of Lj supplementation during pre- and post-natal periods to protect from allergic and viral immune responses in distal mucosal surfaces by altering systemic immune cell responses.

RO1HL144858
ILC3s restrict intestinal inflammation via IL-10 production
Authors: Li Jing , Jacobse Justin , Goettel Jeremy Allen , Washington Mary Kay
Published: August 2023

In this study, we investigated the role of IL-10 production by group 3 innate lymphoid cells (ILC3s) in regulating intestinal immunity. Prior work has established the importance of IL-10 as well as ILC3s in promoting immune homeostasis in the gut. However, the requirement of IL-10 in ILC3s for homeostasis remains unknown.

We examined an ILC3 focused cluster in a publicly available single-cell RNA sequencing dataset from patients with ileal Crohn’s disease and detected ILC3s expressing IL10. We then developed Rag1−/−; Il10GFP; Rosa26lsltdTomato; RorcCre mice and injected them with PBS or anti-CD40 to induce intestinal inflammation and determine if putative ILC3s expressed IL-10. IL-10 was significantly upregulated following treatment with anti-CD40 7 days following disease initiation. To assess whether ILC3 production of IL-10 played a functional role, we targeted IL-10 specifically in ILC3s by generating Rag1−/−; Il10flox/flox; Rosa26lsl−tdTomato; RorcCre (Il10ΔILC3) mice. Il10ΔILC3 mice displayed significantly worse colitis induced by anti-CD40 that was attributed to an increase in Ly6ChiMHCIIhi intestinal macrophages (Mϕ) at baseline. Given the alterations in the phenotype of intestinal Mϕ, we examined whether unfractionated wild-type CD4+ T cells would be sufficient to cause colitis when transferred to Il10ΔILC3 mice despite the presence of regulatory T cells. Mice were monitored weekly for weight loss and colons harvested for histology at 8 weeks. While IL-10-sufficient littermate controls did not develop inflammation as expected, Il10ΔILC3 mice developed severe colitis.

Collectively, our findings identify an essential role for IL-10 production by ILC3s to maintain intestinal immune homeostasis.

Vanderbilt Digestive Disease Research Center Grant (P30DK058404) The National Institute of Diabetes and Digestive and Kidney Diseases R03DK123489
ILC3s select microbiota-specific Treg cells to establish tolerance in the gut
Authors: Lyu Mengze , Suzuki Hiroaki , Kang Lan , Gaspal Fabrina , Zhou Wenqing , Goc Jeremy , Zhou Lei , Zhou Jordan , Zhang Wen ,   JRI Live Cell Bank, Shen Zeli , Fox James G , Sockolow Robbyn E , Laufer Terri M , Fan Yong , Eberl Gerard , Withers David R , Sonnenberg Gregory F
Published: August 2023

Microbial colonization of the mammalian intestine elicits inflammatory or tolerogenic T cell responses, but the mechanisms controlling these distinct outcomes remain poorly understood, and accumulating evidence indicates that aberrant immunity to intestinal microbiota is causally associated with infectious, inflammatory and malignant diseases. Here we define a critical pathway controlling the fate of inflammatory versus tolerogenic T cells that respond to the microbiota and express the transcription factor RORγt. We profiled all RORγt+ immune cells at single-cell resolution from the intestine-draining lymph nodes of mice and reveal a dominant presence of T regulatory (Treg) cells and lymphoid tissue inducer-like group 3 innate lymphoid cells (ILC3s), which co-localize at interfollicular regions. These ILC3s are distinct from but having interconverting potential with extrathymic AIRE-expressing cells, abundantly express major histocompatibility complex class II (MHCII), and are necessary and sufficient to promote microbiota-specific RORγt+ Treg cells and prevent their expansion as inflammatory T helper 17 (TH17) cells. This occurs through ILC3-mediated antigen presentation, αV integrin and competition for interleukin-2. Finally, single-cell analyses suggest that interactions between ILC3s and RORγt+ Treg cells are impaired in inflammatory bowel disease. Our results define a paradigm whereby ILC3s select for antigen-specific RORγt+ Treg cells, and against TH17 cells, to establish immune tolerance to the microbiota and intestinal health.

Crohn’s and Colitis Foundation Research Fellowship Award, 935259
Investigating T cell populations for immune cell exhaustion in myalgic encephalomyelitis/chronic fatigue syndrome
Authors: Maya Jessica , Hanson Maureen R
Published: August 2023

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a devastating disease that confines ~65 million people worldwide to a life of pain, severe exhaustion, cognitive deficits, flu-like symptoms, and postexertional malaise. We previously discovered decreased glycolysis, decreased mitochondrial membrane potential, and increased fatty acid oxidation in ME/CFS T cells. These features are commonly found in exhausted T cells, a reversible state seen in chronic viral infections that decreases cell proliferation, survival, and cytotoxicity. Our study evaluated whether exhausted T cell subsets circulate in people with ME/CFS by analyzing CD4+ and CD8+ T cells for associated inhibitory receptors and transcription factors. CD4+ and CD8+ T cells were isolated from 20 patients and 20 sedentary control blood samples and measured for ~15–17 markers at rest and after stimulation using a FACSymphony analyzer. Markers included cell population identifiers for naïve, effector, memory, regulatory, and helper T cells. We found significantly higher abundances and frequencies of transcription factors related to exhaustion in general and within PD-1+ CD8+ T cells in ME/CFS samples. Half of the ME/CFS cohort showed higher PD-1 memory CD8+ T cell frequencies than the control group. Moreover, ME/CFS CD4+ T cells exhibited higher levels of Lag3, CTLA4, and T-bet in various subsets. This research detects a novel state of immune cell dysfunction in ME/CFS that may suggest new treatment options.

Supported by the Alfred P. Sloan Foundation and the National Institute of Neurological Disorders and Stroke (NINDS), NIH (U54NS105541).
Amplified STAT3 signaling alters expression of CD39 in STAT3 gain-of-function (STAT3 GOF) and in healthy donor CD8+T cells
Authors: Campos Duran Jose S , Sayed Samir , Conrey Peyton E , Hay Ceire A , Dalalo Megan C , Schmitt Erica G , Mauracher Andrea A , Romberg Neil D , Holland Steven M , Freeman Alexandra F , Bergerson Jenna R E , Su Helen C , Leiding Jennifer W , Forbes Lisa R , Cooper Megan A , Vogel Tiphanie P , Henrickson Sarah E
Published: August 2023

It is still unknown whether primary immune regulatory disorders (PIRD) due to genetic variants that amplify T cell receptor and/or inflammatory cytokine signaling yield T cell exhaustion. Here, we investigate the impact of dysregulated cytokine signaling on CD8+ T cells in STAT3 gain-of-function (STAT3 GOF) patients. Immune profiling via 43-parameter CyTOF reveals altered CD8+ T cell differentiation, including loss of naïve cells, increased frequency of effector memory and TEMRA cells, and downregulation of markers of stemness (i.e., TCF-1 and CD127) in STAT3 GOF. Additionally, STAT3 GOF CD8+ T cells demonstrate decreased IL-2 production and proliferation. We identify increased expression of the ecto-enzyme, CD39, which has been reported on exhausted cells and hydrolyzes extracellular ATP, on CD8+ T cells from STAT3 GOF patients and in a mouse model of STAT3 GOF. To identify the signals regulating CD39, healthy donor CD8+ T cells were cultured with STAT3-activating cytokines alone but did not upregulate CD39. However, TCR engagement led to robust CD39 expression which was further augmented with STAT3-activating cytokines. Specifically, CD39 upregulation occurs in pSTAT3Y705+ cells. Finally, sorted CD39+CD8+ T cells hydrolyzed ATP and suppressed effector CD8+ T cell proliferation compared to CD39−CD8+ T cells. These data suggest that STAT3 signaling may play a role in regulating CD39 levels and thus contribute to CD8+ T cell exhaustion, which will be explored in future work. Understanding how dysregulated STAT3 signaling impacts T cell function in these rare patients can improve our understanding of this complex disease and may yield insights into shared aspects of T cell dysfunction found in more common inflammatory diseases.

HHMI Gilliam Fellowship for Advanced Studies (GT15736) Penn Presidential Fellowship PIDTC (Henrickson Lab) IDF (Henrickson Lab) NIH NIAD K08AI135091 (Henrickson Lab) Burroughs Wellcome Fund CAMS (Henrickson Lab)
Dysregulated autophagy in Lymphocytes hints at contribution to immune-mediated thrombotic thrombocytopenic purpura (iTTP)
Authors: Schaller Monica , Milyaev Saveliy , Tschan Mario Paul
Published: August 2023

Immune-mediated TTP is a life-threatening autoimmune disorder characterized by abundant occlusion of the microcirculation. iTTP is caused by a severe autoantibody-mediated deficiency of the von Willebrand factor cleaving protease, ADAMTS-13. Previously, we demonstrated dendritic cells from iTTP patients to have a distinct expression profile of autophagy (ATG)-related genes in 1 relapsing iTTP patient. Contribution of autophagy to the maintenance of autoimmune lymphocytes is crucial to understand the disease pathogenesis of this rare disease.

The autophagy expression profile of purified B- and T-cells isolated from whole blood of iTTP patients or splenocytes using magnetic negative isolation, in the acute or remission phase will be compared to lymphocytes from healthy controls. We choose a panel of nine essential ATG genes (Beclin-1, Atg5, Atg7, Atg14, WIPI1, ULK1, MTOR, NRB1, and WDR45) implicated in other autoimmune diseases to assess a basal level of autophagy by qPCR, autophagic flux (LC3 and p62 expression) will be measured by immunofluorescense. Additionally to discriminate autophagy levels during the acute and relapse phase in relapsing or single episode iTTP patients we will use ImageStream technique.

Preliminary immunofluorescence data from healthy donors indicate that B-lymphocytes show elevated autophagy thanT lymphocytes. These findings are in line with a fact that autophagy participates in antigen presentation and hence may be increased in cells which have antigen presenting function.

Our study will help to shed light on the development and maintenance of iTTP and paves the road to design new treatment strategies based on aberrantly expressed ATG genes by blocking/activating respective autophagy pathways.
Charactering the immune signature of antigen presenting cells in the immune response to factor VIII in a murine model of hemophilia A using single-cell RNA sequencing
Authors: Batsuli Glaivy , York Elizabeth S , Krishnan Upaasana , Gill Surinder , Thomas Beena , Bhasin Swati
Published: August 2023

Deficiency of the procoagulant protein factor VIII (FVIII) is the defining characteristic for individuals with the inherited X-linked bleeding disorder hemophilia A. These individuals require intravenous infusions of FVIII to treat bleeding events, however ~30% develop neutralizing alloantibodies (i.e., inhibitors) against exogenous FVIII. Transcriptome analysis of FVIII-stimulated peripheral blood mononuclear cells in patients with hemophilia A and active inhibitors showed increased expression of innate immune modulators and proinflammatory pathways. Murine models of hemophilia A (FVIII KO) similarly develop anti-FVIII antibodies with FVIII exposure. Although the antibody response to FVIII is CD4-T cell dependent, the key antigen presenting cells (APCs) that mediate inhibitor formation is unclear. Bone marrow chimeras of transgenic mice with depleted dendritic cell (DC) subsets in FVIII KO suggest that type 2 classical DCs (cDC2), but not cDC1 or plasmacytoid DCs, mediate FVIII immunity. We then evaluated the evolution of splenic APCs and their immune cell signatures in FVIII KO mice injected with 0, 1, 3, or 5 doses of FVIII using flow cytometry and single-cell RNA sequencing (scRNAseq). FVIII KO injected with 5 doses of FVIII had similar frequencies of pDC, cDC1, and cDC2 subsets compared to mice injected with normal saline as a control. However, preliminary scRNAseq analysis demonstrated increased expression of phagocytic and interferon-related genes among DC and macrophage clusters, particularly CD8a−CD4+ Itgax− DCs. Further scRNAseq analyses of APC subclusters and differentially expressed genes will be assessed. Overall, these findings suggest that cDCs, specifically cDC2, are critical in FVIII immunity.

Supported by NIH K99HL150595 and Hemophilia of Georgia Clinical Scientist Development Grant.
X-Linked Chronic Granulomatous Disease in a Female Carrier: A Management Challenge
Authors: Sarkaria Sandeep , Hostoffer Robert W
Published: August 2023

Chronic granulomatous disease (CGD) is characterized by recurrent bacterial and fungal infections due to reduced nicotinamide adenine dinucleotide phosphate oxidase resulting in impaired production of reactive oxidant species, and aberrant inflammation. X-linked CGD (XL-CGD) is associated with a mutation in the CYBB gene. XL-CGD affects more males, although some females are affected affected due to skewed X-inactivation. Though management guidelines for CGD patients exist, they remain lacking for clinically affected XL-CGD female carriers. A 3 year old female with history of hematochezia, oral ulcers, and recurrent skin infections who presented with fevers and skin pustules. Pustule cultures were positive for B. cepacian, and colonoscopy was non-acute. Immunology had concern for XL-CGD with recent atypical infections and dihydrorhodamine (DHR) flow cytometry was abnormal, 17.7% (normal greater than 95%). DHR 2 months later showed 13% activation. Genetic testing showed a CYBB, c.45+2dup (intronic), heterozygous, VUS, gene mutation affecting the splice site, associated with XL-CGD, and a NOD2, c.3019dup, heterozygous, with a 2–4 fold increased risk for Crohn’s disease. She was started on bacterial prophylaxis only. She is awaiting gp91phox expression for gene sequencing.

XL-CGD carriers can be at higher risk for infections, and risk is inversely correlated with %DHR neutrophil counts, but only clinically apparent with %DHR less than 20%, and more as levels are less than 10%. Though our patient was started on Bactrim prophylaxis with her %DHR less than 20%, there remains the question of when and if she should be started on anti-fungal prophylaxis. There remain no clear guidelines on the management of XL-CGD carriers.
Identifying immune biomarkers associated with human trauma as potential therapeutic targets
Authors: Fathi Parinaz , Karkanitsa Maria , Rupert Adam , Ricotta Emily ,   Traumatic Injury Drug Prevalence Team, Sereti Irini , Sadtler Kaitlyn
Published: August 2023

Severe trauma can induce systemic immunosuppression, which makes understanding the biomarker profiles of trauma patients critical for therapeutic development and for predicting potential immune responses to treatments such as biomedical implants. Our goal was to establish a data set containing levels of a large array of biomarkers measured in blood from human trauma patients. Multiplex electrochemiluminescence assays were conducted to measure the levels of 59 biomarkers in 1000 blood samples collected from patients at the time of admission to trauma centers. Injury types were categorized as motor vehicle crashes (n=448), falls (n=221), gun wounds (n=78), stabbings (n=117), or other traumatic injuries. Patients ranged in age from 18 to 101, with a mean age of 42.7 for male patients (n= 725) and 49.8 for female patients (n= 272). There were no significant differences in biomarker expression levels between the different injury types. Differences in age distributions of male and female patients for each injury type were not found to be statistically significant, except in the case of falls, which had a higher average age for female patients compared to male patients. MIF, MIP-5, and YKL-40 had the highest expression, with concentrations of 554148 ± 62938 pg/mL, 17409 ± 919 pg/mL, and 98793 ± 10015 pg/mL (mean ± 95 % CI) respectively, at 1:100 dilution. Further experiments are currently being conducted to establish biomarker profiles for control uninjured human samples, and data comparing the profiles of trauma patients and uninjured controls will be presented. Overall, this work provides a resource for understanding immune responses to injury and potentially developing new therapeutics with these biomarker profiles in mind.

This work was supported by the National Institute of Biomedical Imaging and Bioengineering
Analysis of novel RAC2 variants associated with neutrophil dysfunction, lymphopenia, and primary immunodeficiency
Authors: Donko Agnes P , Sharapova Svetlana O , Marois Louis , Winterbourn Christine , Ashby Louisa , Martelius Timi , Seppänen Mikko , Andreae Doerthe A , Leiding Jennifer W , Gauthier Amélie , Campbell Nicholas , Ganesan Sundar , Hsiao Kuang-Chih , Holland Steven M , Falcone Emilia L , Hsu Amy P , Leto Thomas L
Published: August 2023

RAC2 is a member of Rho-family GTPases, abundant in neutrophils serving roles in NADPH oxidase activation and cytoskeleton dynamics. Patients with dominant, activating mutations in RAC2 (E62K, N92T, Q61R) are associated with neutrophil dysfunction, lymphopenia and immunodeficiency. We now characterize additional disease-related RAC2 variants with in-vitro analysis of RAC2 effector functions including NOX2-based superoxide production, regulation of PAK1-PDB binding, activation of AKT, protein stability, subcellular localization, and F-actin formation.

We identified both active and inactive variants associated with lymphopenia and immunodeficiency. Patients with dominant, active RAC2 variants with high protein stability (Q61R, Q61K) presented in the first days of life with severe combined immunodeficiency and absent lymphocytes. Activating variants with lower protein stability (I21S, E62K, N92K, N92S) presented as combined variable immunodeficiency with lymphopenia, detectable by newborn screening but clinically presenting later with recurrent sinopulmonary infections, reduced B-cells and hypogammaglobulinemia. A third class of mutations with unstable transcript or protein were phenotypic only in homozygosity (W56*, R68W). Severity of phenotype and initial clinical presentation was correlated with protein stability, with increased stability leading to neonatal presentation and decreased stability of dominant mutations having later clinical onset. Thus, heterologous expression and analysis of RAC2 variants is useful for deciphering the genetic and molecular mechanistic basis for the common underlying clinical phenotype related to lymphopenia and immunodeficiency.
Generation of thymic epithelial progenitor cells from induced pluripotent stem cells obtained from patients with thymic defects
Authors: Amini Kayla , Pala Francesca , Bosticardo Marita , Notarangelo Luigi D
Published: August 2023

Thymic epithelial cells (TECs) play a critical role in T-cell development as they provide essential cues to hematopoietic progenitors for their proliferation and differentiation in the thymus. Patients with genetic mutations affecting TEC functionality present with reduced thymus development and decreased T-cell output in the periphery. We have previously reported that T-cell development is blocked in patients with hematopoietic-intrinsic genetic defects using an artificial thymic organoid system, but this model does not allow testing of TEC defects. To address this, we have generated multiple induced pluripotent stem cell (iPSC) lines from patients carrying mutations on genes involved in thymic stromal development and/or function, including 22q11.2del (DiGeorge syndrome), CHD7, FOXI3, PAX1, AIRE, EXTL3, and TP63. We aim to differentiate these iPS lines into TECs and assess whether these mutations alter TEC generation. We are currently testing previously published protocols. However, most of these protocols focused on generating TECs from embryonic stem cells and were not optimized for human iPSCs. To address this, we are comparing and combining different methods to achieve a high efficiency in inducing definitive endoderm (DE), ventral pharyngeal endoderm (VPE) and finally thymic epithelial progenitors (TEP). We have already successfully reached the DE stage, as gene expression analysis by qPCR shows upregulation of genes associated with the DE and downregulation of iPS genes. Once we successfully generate TECs, we plan to incorporate them into our artificial thymic organoid system to create a model that will be solely based on human cells, obtaining a more accurate in vitro reflection of human thymic T cell development.

This work was supported by the Division of Intramural Research (DIR), NIAID, NIH
IL-21 or synthetic IL-2 treatment drives T cell IFNγ production or expansion, respectively, during adoptive cell therapy of melanoma
Authors: Walsh Michael , Zhangxu Kevin , Lim Birkley , Liu Samantha , Kureshi Rakeeb , Dougan Stephanie , Dougan Michael
Published: August 2023

Adoptive cell therapy (ACT) of tumor-infiltrating lymphocytes (TILs) can generate long-term durable responses in patients with melanoma and can even successfully treat patients that are refractory to anti-PD-1 checkpoint blockade. IL-2 is often used to expand TILs, however the administration of this cytokine can induce systemic toxicities in patients. Synthetic cytokine mimetics of IL-2 have been developed, including neoleukin-2 (Neo2), which has been shown to have enhanced therapeutic efficacy over IL-2, with reduced toxicity. IL-21 has also been used in TIL therapy to promote T cell expansion. Here, we tested the efficacy of Neo2 or IL-21 in treating mice bearing B16 tumors that received ACT with melanoma-specific T cells (TRP1High and TRP1Low; TCRs recognizing antigen with high or low affinity). Both Neo2 and IL-21 could slow growth of B16 tumors, curing several mice. We performed single-cell RNA-sequencing on these tumors to analyze cytokine therapy-induced changes in the microenvironment. Neo2-treated tumors had a signature primarily of T cell expansion, with few other associated transcriptional changes. IL-21 therapy generated a profound IFNγ signature, with an increase in T cells production of IFNγ and many cell types, including T cells, macrophages, and tumor cells responding with interferon-stimulated gene expression. Interestingly, IL-21 therapy did not induce profound T cell expansion as Neo2 did, but importantly both IL-21 and Neo2 therapy reduced levels of Tregs within the tumor. IL-21 also improved T cell production of granzyme B. Thus, IL-2 primarily expands T cells and IL-21 generates higher quality anti-tumor T cells. Therefore, IL-21 is a promising cytokine for improving ACT and other immunotherapies.
Overcoming immunotherapy resistance in melanoma defective of IFN-γ signaling
Authors: Shi Lewis Zhichang , Shen Hongxing , Ojo Gbenga , Bonner James A
Published: August 2023

Therapeutic resistance to immune checkpoint blockers (ICBs) in melanoma patients is a pressing issue. Initial efforts from others and us show that ICB resistance can derive from melanoma-intrinsic loss of the IFN-γ signaling, which occurs in ~20.9% of the 287 melanoma cases (CBioPortal), based on the cumulative frequency of damaging mutations of 12 core genes. However, how to overcome this resistance mechanism remains largely unexplored. Moreover, given the indispensable role of tumor-infiltrating T cells (TILs) in ICBs, little is known about how tumor-intrinsic loss of IFN-γ signaling (IFNγR1KO) impacts TILs. Here, we report that IFNγR1KO melanomas have reduced infiltration and function of TILs. Combining kinomics, RNA-seq, and proteomics, we unveil that Janus Kinase 1 and 2 (JAK1-2), key downstream kinases in the IFN-γ signaling cascade, are constitutively active, mediated by heightened mTOR. Importantly, mTOR inhibition with Everolimus induces a potent suppression of IFNγR1KO melanoma growth, particularly when combined with ICBs, despite its reported modest effects on WT tumors; similarly, targeting JAK1-2 with Ruxolitinib (Ruxo) selectively impedes the growth of IFNγR1KO but not WT melanomas, revealing effective “targeted” treatments for ICB resistance. Mechanistically, these therapeutics successfully reprogram “cold” TILs in IFNγR1KO melanoma to functional effectors (e.g., increased production of TNF), and deleting T cells or using TNFKO mice completely abrogates Ruxo efficacy. Altogether, our results highlight an active role of tumor-intrinsic IFN-γ signaling in shaping TILs and manifest therapeutic targets that can be harnessed to bypass ICB resistance of melanomas defective of IFN-γ signaling.

Supported by grants from NIH (1R21CA259721-01A1) and from DoD Idea Award (ME210108)