bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of regulatory natural killer cells in pediatric chronic graft-versus-host disease patients
Authors: Mei Ao , Lauener Madeline P , Schultz Kirk R , Malarkannan Subramaniam
Published: August 2023

Chronic graft-versus-host disease (cGVHD) is the leading cause of late morbidity and mortality in pediatric patients, which is caused by dynamic interferences of multiple types of immune cells, including natural killer (NK) cells. While earlier studies have indicated the potential regulatory function of NK cells restraining pathogenic cell types, the identity of unique NK cell subsets remains unknown. We performed single-cell RNA sequencing on NK cells isolated from the PBMC of patients who developed cGVHD (3 patients) or not (4 patients). Through computational analyses, we identified distinct NK cell populations and found multiple subsets of NK cells expressing unique transcriptomes in patients who received hematopoietic stem cell transplantation (HSCT) compared to healthy individuals, with one of the NK cell subsets exclusively present in post-HSCT patients. Significantly different gene expression patterns were observed between the two groups of patients, including cytotoxicity-related genes elevated in cGVHD patients. By applying diverse statistical analyses, we were able to characterize these NK cell subsets, indicating their potential roles in the development or prevention of cGVHD. Our study defines novel subsets of protective NK cells during cGVHD. These findings are of high translational relevance and can contribute to the treatment of cGVHD patients.
Immune-Modifying Microparticles for Treatment of Acute Graft-Versus-Host-Disease
Authors: Beddow Sara A , Lust Hannah , Galvin John , Miller Stephen D
Published: August 2023

Background

Acute Graft versus host disease (aGVHD) is one of the most common complication of allogeneic hematopoietic stem cell transplant. We have previously shown that infused immune-modifying particles (IMPs) can reduce clinical symptoms and improve survival in inflammatory conditions. IMPs are taken up by inflammatory monocytes (θIM) via the macrophage receptor with collagenous structure (MARCO) and then undergo splenic sequestration. Given these findings, IMPs may be an effective method of reducing inflammation in aGVHD.

Methods

We tested the efficacy of IMP in an established aGVHD murine model. We also explored the changes in immune cell subsets and serum cytokines. To assess impact of IMP on graft-versus-tumor (GVT), we infused host mice with A20 lymphoma cells.

Results

We demonstrated that treatment with IMPs effectively rescues mice in a lethal aGVHD model. Treatment with IMPs also significantly improved clinical and target organ histopathological scores. IMP treatment resulted in increased splenic and intestinal CD4+CD25+Foxp3+ Tregs. IMP treated mice had lower numbers of colonic CD11b+Ly6chi θIM and a lower peak of serum inflammatory cytokines. Maintenance of GVT effect was demonstrated by comparable survival rates in host mice with co-infusion of A20 lymphoma cells.

Conclusion

Systemic IMP was effective at rescuing mice in this lethal aGVHD model. This demonstrates that targeting θIMs with IMPs could be an effective strategy to treat aGVHD. Further investigation of the mechanisms by which IMPs ameliorate aGVHD is warranted, as is investigation of the use of IMPs to treat aGVHD in the clinical setting.

Supported by Nanoparticle Tolerance Research Fund
Immune profile changes induced by extracorporeal photopheresis as prophylactic therapy in lung transplanted cystic fibrosis patients
Authors: Fenizia Claudio , Righi Ilaria , Vanetti Claudia , Biasin Mara , Rosso Lorenzo , Torretta Lorella , Mocellin Maria Cristina , Morlacchi Letizia , Rossetti Valeria , Diotti Cristina , Nosotti Mario , Clerici Mario , Trabattoni Daria
Published: August 2023

Chronic rejection (CR) is the leading cause of late morbidity and mortality upon lung transplantation (LuTx). Extracorporeal photopheresis (ECP) has emerged as a promising immunomodulatory prophylactic induction treatment against rejection. We performed and in-depth investigation of the immunological effects of ECP in LuTx recipients with a diagnosis of cystic fibrosis (CF).

A pilot clinical trial enrolled 20 CF LuTx patients who were randomly allocated in 2 arms: standard immunosuppressive therapy alone vs. standard immunosuppressive therapy plus ECP for 3 months, beginning within 72h from LuTx. Functional activation of T and NK subpopulations as well as mRNA expression and cytokine secretion profiling were evaluated in peripheral blood and in bronchoalveolar lavage (BAL) up to 12 months after LuTx. Clinical parameters, including respiratory volumes (e.g. FEV1), rejection episodes and infections, were analyzed as well.

ECP was well tolerated with no complications nor opportunistic infections. Rejection rate was comparable in the two groups Notably, a significantly better FEV1 was observed in the ECP group overtime. Treg lymphocytes and IL10-producing NKs were significantly increased, while Th17 cells were significantly reduced in the ECP group compared to the control. Cytokine profile showed that ECP reduced pro-inflammatory cytokines (e.g. IL1b, IL6) production, increasing that of anti-inflammatory cytokines (e.g. IL10, IL1RA) both in plasma and BAL

ECP is associated with immune modulation resulting in improved patients’ respiratory performance. More extensive studies and longer follow-up are needed to verify if ECP-induced immune modulation will have a beneficial effect of organ rejection as well.

Supported by grants from the Cystic Fibrosis Foundation
Dietary fat source induces gut microbiota alterations and impacts chronic murine graft- versus-host disease severity
Authors: Millick Danielle , Strair Roger , Sant’Angelo Derek
Published: August 2023

It has been observed that a constriction of the gut microbiome occurs with high fat diet, and likewise decreased microbiome diversity is directly correlated with more severe graft-versus-host disease (GvHD) following allogeneic hematopoietic stem cell transplantation. Taken together it is plausible that not only fat content, but source of dietary fat could influence gut microbiota and GvHD outcome. We sought to address this question and developed experimental rodent diets that have the same fat content (25% kcal fat, a moderate increase in fat content from normal chow) which differ only by fat source: animal- versus plant-derived fat and found that these diets drive alterations in gut microbiota diversity. Both diets yielded similar outcomes in an acute model of GvHD, however we found that mice fed the animal-derived fat diet exhibited worse survival and outcomes in a model of chronic GvHD, than mice fed the plant-derived fat diet. These findings correlated with increased inflammation and alterations in intestinal immune cell populations following chronic GvHD induction. Collectively, our data show that diets that are equal in macronutrient content but different in fat source can promote different gut microbiomes and in turn alter intestinal homeostasis and chronic GvHD outcome.

Century for the Cure - Philanthropic Fund
Examining the Relationship Between Gamma Delta T Cells and Sensory Peripheral Nerves in the Skin
Authors: Almaguer Berenice , Tulloch Patricia , La Prairie Chloe , Jameson Julie M
Published: August 2023

Wound repair involves a complex system of immune cells and mechanisms to induce proper healing of a laceration. The transmission of pain signals and immune functions occur simultaneously, suggesting a collaboration between immune and nervous systems. Gamma delta (γδ) T cells are present in the epidermis and dermis where they participate in wound repair by producing growth factors, cytokines, and chemokines. Dermal γδ T cells are adjacent to nerves in the skin. We set out to determine whether sensory peripheral nerve endings and γδ T cells exhibit crosstalk during wound repair. Using immunofluorescent microscopy we found that peripheral neurons and γδ T cells are in close proximity in both the epidermis and the dermis suggesting a transmission of signals. To determine whether γδ T cell dysfunction impacts neurons, we examined non-wounded and wounded skin derived from lean versus diet-induced obese (DIO) mice. There was less interaction between γδ T cells and sensory peripheral nerves in both nonwounded and wounded DIO mouse skin. To determine whether the lack of γδ T cells impacts peripheral nerve repair and wound healing responses, we compared wildtype and T-cell receptor knockout (TCRd−/−) mice. TCRd−/− mice did not exhibit any differences in the number of sensory peripheral nerves in nonwounded skin. However, there were fewer sensory peripheral nerves surrounding the wound edge in mice lacking γδ T cells. Together this data indicates that γδ T cells regulate how sensory peripheral nerves innervate a wound, but are not required for neuronal development. Understanding the mechanism that regulate the interdependent relationship between γδ T cells and sensory peripheral nerves could further our understanding of how to improve wound repair.
IL-35 Restricts Serine Metabolism to Suppress Effector T Cell Function in Atopic Dermatitis
Authors: Choi Jin Kyeong
Published: August 2023

IL-35 is an immunosuppressive cytokine and a critical regulator of immunity in autoimmune diseases. However, whether IL-35 controls metabolism to inhibit T cell development in atopic dermatitis (AD) remains unclear. In this study, we demonstrated that IL-35 ameliorates AD by inhibiting the serine metabolic enzyme, phosphoserine aminotransferase 1 (PSAT1), in mice and humans. When the major metabolic pathway was assessed, IL-35 was found to inhibit increased serine metabolism in activated T cells. Measure of the ear thickness, histological examination showed that treatment of the mice with IL-35 inhibited AD inflammation by suppressing expansion of Th1, Th2, Th17 cells while inducing expansion of Foxp3-expressing Tregs. IL-35 also regulated alterations in glycolytic metabolism in mice and patients with AD. Importantly, the IL-35-mediated suppression of Th2 cells and amelioration of AD correlated with the inhibition of serine metabolic pathways, such as PSAT1, in the skin. Herein, IL-35 was found to inhibit effector T cells in AD by directly regulating the serine metabolic enzyme, PSAT1. Therefore, therapies that target serine-dependent metabolic pathways via IL-35 could serve as effective therapeutic strategies for AD.
Single cell transcriptomic changes of CD8T cells in male and female patients with coronary artery disease and diabetes
Authors: Iqneibi Shahad , Saigusa Ryosuke , Suthahar Sujit Silas Armstrong , Khan Amir , Oliaeimotlagh Mohammad , Gulati Rishab , Suryawanshi Vasantika , Alimadadi Ahmad , Pandori William , Makings Jeff , Durant Christopher , Ghosheh Yanal , Durant Fabrizio , McNamara Coleen , Shemesh Avishai , Lanier Lewis L , Hedrick Catherine , Ley Klaus
Published: August 2023

Objective

The objective of this study was to determine the role of CD8+ T cells in subjects with coronary artery disease (CAD) with and without diabetes (DM).

Methods

PBMCs of 60 men and women who underwent cardiac catheterization were interrogated by single cell RNA sequencing combined with 49 protein markers (CITE-Seq). Four pairs of groups were matched for all clinical and demographic parameters.

Results

140,610 single cell transcriptomes were successfully analyzed. Among them, 17,349 CD8+ T cells were identified as CD19− CD3+ CD4− CD8+, yielding 17 clusters. The proportion of cells in the CD137+ effector memory (EM) cluster were significantly decreased in CAD+ subjects when compared to matched CAD− subjects. This same cluster was significantly decreased in DM+ CVD− groups when compared to the matched DM− CVD− group. The NK-like Emra1 group was significantly more abundant in CVD+ subjects and in females compared to males. One of the regulatory CD8 T cell clusters was significantly reduced in diabetics. 30 genes were significantly upregulated in both CAD and DM. The DM gene signature was partially additive to the CAD gene signature. IFTM3 and RUNX3 showed more importance in females, TCF7 and LTB in males.

Conclusions

We conclude that CAD is clearly reflected in PBMC transcriptomes and that significant differences exist in immune cell transcriptomes between women and men and between diabetic and non-diabetic subjects.

Support by the NIH P01 HL 136275
Regulatory T cells in the fibrotic milieu: ontological characterization from initiation through remodeling in a murine model of spontaneous pulmonary fibrosis
Authors: Murthy Aditi , Rodriguez Luis , Tomer Yaniv , Iyer Swati , Chavez Katrina , Dimopoulos Thalia , Katzen Jeremy , Beers Michael
Published: August 2023

Using exogenous injury models, regulatory T cells (Tregs) have been shown to have both tissue protective (immunosuppressive) as well as deleterious (pro-fibrotic) effects on the lung. The goal of this study was to define the ontogeny of Tregs in a disease relevant preclinical model of spontaneous pulmonary fibrosis (PF). Transgenic Treg reporter and delete mouse lines were generated by crossing the published PF model, IER-SP-CI73T, to Foxp3-GFP or Foxp3-DEREG lines. Tamoxifen induction in IER-SP-CI73T mice resulted in a disease phenotype characterized by 3 phases of disease development corresponding to early initiation (7d), peak inflammation (14–21d) and fibrogenesis (28–42d). During resolution of inflammation and transition to fibrogenesis, we noted an increase in Tregs measured as either expression of CD25hiFoxP3+ or CD4+GFP+T cells at 21–28 days after Sftpc induction in either model. IHC demonstrated FoxP3+ cells localized to perivascular regions. Administration of diphtheria toxin to IER-SP-CI73T/Foxp3-DEREG mice at 17 days resulted in systemic loss of Tregs and increases in morbidity (weight loss), BALF cell counts, and tissue neutrophils accompanied by reduced lung compliance. RNA-seq of bulk isolated GFP+ (Treg) cells from the fibrotic phase (Day 21) identified enhanced proliferation signals. Collectively, the data demonstrate a clear pattern of Treg recruitment coinciding with the transition of inflammation resolution to fibrogenesis and suggest an essential protective role for Tregs through a dampening of underlying inflammation in pulmonary fibrosis.

Supported by NIH 2R01 HL 095993-10
Identified the pathogenic effects of GM-CSF autoantibodies in different diseases
Authors: Lo Yu-Fang , Wang Shang-Yu , Shih Han-Po , Chung Pei-Han , Ku Cheng-Lung
Published: August 2023

Pathogenic GM-CSF autoantibodies are found in over 90% of people with pulmonary alveolar proteinosis (PAP), a rare pulmonary autoimmune disease with surfactant lipoprotein accumulating in alveoli spaces. However, more and more people with GM-CSF autoantibodies have also been identified without PAP phenotype but underlying severe central nervous system (CNS)-involved Cryptococcus gattii or Nocardia spp. infection since 2013, highlighting the pathogenic mechanisms that might differ between individual diseases.

We developed single B cell cloning to isolate GM-CSF monoclonal antibodies from cryptococcosis patients aiming to investigate the pathogenic effects. GM-CSF autoantibodies in cryptococcosis bound GM-CSF through three non-overlapping epitopes, and formed a high-molecular-weight immune complex similar to the research of GM-CSF mAbs in PAP patients. Surprisingly, GM-CSF mAbs isolated from cryptococcosis revealed potentially binding to GM-CSF when the V gene reversed to the germline. GM-CSF autoantibodies underwent somatic hypermutation to enhance their binding ability and neutralizing activities. We analyzed whether the mAbs binding sites interfere with cytokine-specific receptor interaction, and only one epitope showed the receptor blockade. The GM-CSF mAbs from the other two epitopes remained inhibition effects, indicating another neutralizing mechanism independent of interfering receptor binding. The neutralizing pathway and biological impact of GM-CSF autoantibodies from cryptococcosis is still worthy of further investigation. These works might help know the anti-GM-CSF autoantibodies cause different clinical manifestations and find a novel pave for better diagnosis and treatments.
The effects of S100A8/A9 blockade in experimental Myocardial Infarction detected by CITE-seq RNA sequencing
Authors: Vallejo Jenifer , Jakobsson Gabriel , Ljungcrantz Irena , Schiopu Alexandru
Published: August 2023

Background

The alarmin S100A8/A9, is released from neutrophils in response to inflammatory stimuli, but monocytes, and macrophages can also secrete extracellular S100A8/A9. During the acute phase of myocardial infarction (MI), S100A8/A9 is released from the site of the ischemic injury and increases in the coronary and systemic circulation. We have previously shown that S100A8/A9 plays an important role in the inflammatory and reparatory immune responses in MI, and that S100A8/A9 blockade improves cardiac function post-MI.

Methods

In the present study we aimed to understand, on local and systemic level, the influence of S100A8/A9 blockade, by sequencing 30,000 cells by single-cell RNA (scRNA-Seq) and CITE-Seq. MI mice were induced by permanent coronary artery ligation, followed by the S100A8/A9 blocker ABR-238901 or PBS for 3 days. CD45+ cells were isolated from blood, heart, and bone marrow.

Results

Cell populations in blood generally reflected the changes induced by MI in the bone marrow and heart, but we also detected important shifts specific for the MI. ABR-238901 reduced the expression of pro-inflammatory genes and limited inflammatory neutrophil and monocyte sub-populations in blood and heart. In bone marrow the treatment inhibited the expression of genes involved in proliferation and differentiation of hematopoietic stem cells.

Conclusions

S100A8/A9 blockade has potent immunomodulatory effects in MI through inhibition of myelopoiesis and population shifts from pro-inflammatory to reparatory neutrophils, monocytes and macrophages in blood and heart. We have also identified pathogenic sub-populations in the ischemic heart that could be therapeutically targeted to reduce myocardial inflammation and damage.
CCR10 promotes blood pressure elevations and renal damage in a mouse model of hypertension
Authors: Nguyen Bianca A , Chen Ziche , Fehrenbach Daniel J , Zhong Xue , Xiong Na , Madhur Meena , Alexander Matthew R
Published: August 2023

Hypertension is the leading cause of morbidity and mortality worldwide. Emerging evidence suggests that pro-inflammatory immune cells play a central role in hypertension and its associated end-organ damage. Regulatory T cells (Tregs) are immunosuppressive, however their role in hypertension remains unclear. Interestingly, Tregs can be pathogenic in some disease states by limiting angiogenesis. We recently identified a selective decrease in circulating CCR10+ Tregs in hypertensive humans. CCR10 is a chemokine receptor important for recruitment of immune cells, particularly Tregs, to the skin. Prior studies have demonstrated skin microvascular rarefaction in hypertension as a mechanism to raise blood pressure (BP). We hypothesized that CCR10+ Tregs promote hypertension through skin infiltration leading to enhanced microvascular rarefaction. We demonstrate that mice with angiotensin II (Ang II) infusion to induce hypertension have an increased abundance of CCR10+ Tregs in the skin compared to normotensive controls. In addition, mice with CCR10 deficiency exhibit increased CD31+ endothelial cells in the skin after Ang II infusion compared to wild type controls, consistent with reduced cutaneous microvascular rarefaction. Moreover, CCR10-deficient mice have lower Ang II-induced increases in systolic BP and lower levels of albuminuria. Finally, using PrediXcan, we demonstrate that higher genetically predicted levels of CCR10 are associated with increased risk of hypertension in humans. Taken together, our data suggest that CCR10 promotes Ang II-induced BP elevations and renal damage in mice at least in part by promoting Treg recruitment to the skin leading to microvascular rarefaction.
Tunable dynamics of B cell selection in gut germinal centers
Authors: Nowosad Carla R , Mesin Luka , de Castro Tiago Rezende , Wichmann Christopher , Donaldson Gregory , Lockhart Ainsley , Bilate Angelina , Mucida Daniel , Victora Gabriel
Published: August 2023

B lymphocytes are central to maintaining balanced enteric responses through production of immunoglobulins (Igs). A large fraction of Igs are produced in germinal center (GC) responses, which form upon immunization or infection in the majority of secondary lymphoid organs, but are constitutively present in gut-associated lymphoid tissue. However, the ontogeny and antigenic drivers of constitutive gut-associated GCs were poorly understood. Using a multicolor fate-mapping approach with the Confetti fluorescent allele to visualize GC clonal expansions, we observed single-color GCs in the steady state intestine, suggestive of strong selection events. By coupling fate-mapping, gnotobiotics and Ig sequencing, we showed affinity-driven selection occurring in gut-associated GCs in response to members of the microbiome. We observed that these GCs persist even in the absence of microbiota, although their isotype distribution and clonal composition are altered. Furthermore, we identified several prominent public Ig clonotypes in intestinal GCs, whose presence were tuned by microbial diversity.
IgA deficient mice develop a gluten sensitive enteropathy specifically in the ileum
Authors: Ball Ryan A , Mohamed Ahmed , Kubinak Jason
Published: August 2023

Secretory IgA is the most abundant antibody in the intestinal mucosa and functions as the first line of defense against pathogens and micro-organisms in the gut. Selective IgA deficiency is the most common form of antibody deficiency. Clinically, IgA deficient patients are largely asymptomatic, they are however at a higher risk of developing inflammatory conditions like Celiac disease; an SI-specific enteropathy triggered by exposure to dietary gluten. Interestingly, celiac patients are 10–15 times more likely to have an IgA deficiency. Recent studies have shown that IgA−/− mice develop enteropathy specifically in the ileum. We confirmed this phenotype in our IgA−/− mice and then asked the question; are IgA−/− mice sensitive to gluten? To answer this question, we singly housed IgA−/− mice on a gluten free or nutritionally matched gluten rich diet for 4 weeks and quantitatively assessed their small intestine for histopathological changes. We found that IgA−/− mice on a gluten rich diet develop a significant shortening of villi in their ileum compared to mice on a gluten free diet. Next, in order to exacerbate the phenotype, we developed a line of IgA−/− mice which had never been exposed to gluten and After 8 weeks of age, challenged these mice with 10 mg of concentrated gluten or sterile water every other day for 3 weeks. We found that mice challenged with gluten developed significant shortening of their villi compared to the control. We then used high parametric flow cytometry to compare these findings to shifts in the immunological tone of the SI. Results from our experiments indicate that IgA−/− mice will be a useful model for downstream studies on the mechanisms driving enhanced risk for developing gluten sensitivity in IgA-deficient humans.
Premature Treg induction in early life results in FoxP3 instability post-weaning
Authors: Cheifetz Talia Raitt , Greenfield Kara G , Knoop Kathryn A
Published: August 2023

Peripherally-derived RORγt+ regulatory T cells (Tregs) induced in early life are crucial for maintaining oral tolerance later in life, and the loss of these long-lived cells is associated with increased allergic and inflammatory phenotypes in several animal models. We have identified a predominant pathway of luminal antigen delivery that is associated with Treg development. Around weaning, microbial and dietary proteins are delivered to the colonic lamina propria via goblet cell-associated antigen passages (GAPs) and taken up by antigen presenting cells. GAP formation is inhibited by breastmilk-derived epidermal growth factor (EGF) which is highest in maternal milk at parturition and is diminished by weaning. We have previously shown that disruption of this process – via EGF receptor inhibition (EGFRi) day of life (DOL)4–8 or by conditional deletion of EGFR from goblet cells – results in premature antigen delivery. Consequently, this results in an early initial increase in FoxP3 expression in the colon that is unstable and, ultimately, lost by Tregs primed pre-weaning; this suggests that earlier education of T cells is incompatible with regulatory T cell differentiation in early life. EGFRi-treated FoxP3eGFP-Cre-ERT2ROSAlsl-YFP mice, whose FoxP3+ cells were YFP-labeled at DOL21, had a larger number of YFP+ T cells that lost FoxP3 expression than that of control mice, by DOL28. Further, compared to controls, wild-type mice treated with EGFRi DOL4–8 had increased IFNγ, TNFα, and IL-17a-expressing CD4 T cells when challenged with DSS colitis later in life. This suggests that early antigen delivery-associated FoxP3 instability may contribute to an increased propensity toward an inflammatory phenotype later in life.
Regulating the Regulators: determinants of lineage- and tissue-specific Il10 expression in CD4+ T cells
Authors: Hegner Courtney Lynn , Chen Mei Lan , Sundrud Mark S
Published: August 2023

IL-10 is an essential anti-inflammatory cytokine involved in mucosal immune regulation. Whereas many lineages of innate and adaptive immune cells can produce IL-10, a comprehensive understanding of the common, lineage-specific and tissue-specific regulatory mechanisms underlying Il10 transcriptional regulation has remained elusive. Our lab recently identified the constitutive androstane receptor (CAR; encoded by Nr1i3)—a xenobiotic-sensing nuclear receptor (NR) with no previously known function in immune cells—as a selective regulator of Il10 gene expression in small intestinal (SI) type 1 regulatory (Tr1) cells. CAR induction of IL-10 expression in SI Tr1 cells stands in direct contrast with orthogonal mechanisms that underlie Il10 gene regulation in large intestinal (LI) Foxp3+ T regulatory (Treg) cells, and that involve other nuclear receptors, such as vitamin D receptor (VDR). By using a combination of next-generation sequencing techniques (ATAC-seq, CITE-seq, ChIP-seq), I am assessing how, where and in which cell types CAR most prominently regulates Il10 expression in vitro and in vivo. Our results are generating high resolution maps of Il10 transcriptional regulatory networks that are differentially called upon in different cell types and discrete mucosal tissues to achieve the complex and dynamic regulation of Il10 expression that is observed in vivo.

Supported by grants from NIH (RO1 AI143821-03)
Defining the abundance, fate, and function of secondary lymphoid organ resident memory T cells
Authors: O’Flanagan Stephen D , Künzli Marco , Scott Milcah , Gavil Noah Veis , Quarnstrom Clare F , Smith Olivia , Jackson Julia , Vezys Vaiva , Masopust David
Published: August 2023

For sixty years we have understood that lymphocytes recirculate through blood and secondary lymphoid organs (SLO). Recently, it has been observed that SLOs contain resident populations of memory T cells. Here, we show that conventional isolation techniques grossly underestimate the proportion of SLO CD8+ TRM, and resident cells constitute a substantial fraction of regionalized immunity within LNs. We found that SLO TRM are very long-lived in mice and persist for >500 days after a single infection with LCMV Armstrong. Using a variety of infection models, we observed that the location of primary infection biased the density of SLO TRM to specific draining LNs. Moreover, SLO TRM expressed a phenotypic relationship with TRM in specific upstream nonlymphoid tissues, and remarkably, retained durable expression of regional homing markers. Upon reactivation, mesenteric LN derived SLO TRM preferentially homed to the small intestine compared to TCM. Thus, TRM comprise a substantial fraction of LN memory CD8+ T cells, are refractory to isolation, can be remarkably durable, retain tissue-specific homing potential in the event of reactivation, and likely comprise an underappreciated and underestimated contributor to LN immune surveillance and regional anamnestic immune responses.

Supported by fellowship from the University of Minnesota - Department of Microbiology and Immunology Dennis W. Watson Fellowship
Adaptive immunity to ancient retroelements controls the skin tissue threshold of activation
Authors: Wells Alexandria , Lima-Junior Djalma S , Link Verena M , Krishnamurthy Siddarth R , Belkaid Yasmine
Published: August 2023

A symbiotic dialogue between the microbiota and host is essential to promote protective immune responses in the skin. Integrated retroviral elements, or retrotransposons (RTNs), comprise up to 45% of the human genome. We previously found that immune responses to the skin microbiota are dependent on the reactivation and keratinocyte-intrinsic sensing of RTNs. Here, we explored the possibility that RTNs may also represent a source of antigens recognized by the skin adaptive immune system. We first established a model of sterile injury, where topical application of a mild detergent reactivated RTNs, and recruited CD8 T cells to the skin in an RTN-dependent manner. RNAseq analysis of RTN expression revealed Langerhans cells (LCs) have the most abundant RTN expression amongst skin dendritic cells. Our work demonstrated that CD8 T cell responses to sterile injury were entirely dependent on LCs, supporting the idea that LCs could present RTN antigens to CD8 T cells. To identify RTN-specific T cells, we selected peptides that were uniquely expressed in LCs, but not in thymic epithelial cells which mediate negative selection. Several peptides promoted cytokine production from injury-elicited CD8 T cells, confirming the presence of RTN-reactive CD8 T cells in the skin. RNA-seq analysis suggests RTN-specific CD8 T cells may contribute to wound repair and immunoregulation. Thus, mild stress, such as that induced by mild detergent, is sufficient to promote adaptive immunity to defined antigens derived from RTNs. Our work also proposes that homeostatic immunity to ancient retroviruses promotes a defined class of T cells responses aimed at maintaining barrier integrity in response to daily stressors encountered by the skin.

Cancer Research Institute, Irvington Postdoctoral Fellowship
C. difficile infection induces toxin A specific responses
Authors: Maslanka Jeffrey , Denny Joshua E , Alam M D Z , Alameh Mohamad-Gabriel , Zackular Joseph P , Abt Michael C
Published: August 2023

The nosocomial pathogen Clostridioides difficile colonizes the gastrointestinal tract of patients following microbiome disruption and secretes two major virulence factors toxin A (TcdA) and toxin B (TcdB). Approximately 25% of patients infected with C. difficile experience recurrent infection, and the rate of recurrence increases with each subsequent infection. The quality and protective capacity of the adaptive immune response generated following infection is not well defined. Using a mouse model, we saw high IgG and IgA antibody titers in the serum and stool to TcdA following C. difficile infection. Further, to identify toxin specific CD4+ T cells, a peptide library was generated from the amino acid sequence of TcdA and subsequently used to test for peptide reactive T cells. CD4+ T cells from infected mice produced IL-17a following restimulation with TcdA peptides, with the number of peptide reactive CD4+ T cells correlating with TcdA specific antibody titers. Furthermore, cellular responses were confined to the gastrointestinal tract, with expansion of germinal center B cells, T follicular helper cells, and TcdA specific CD4+ T cells in the lamina propria and mesenteric lymph nodes, but not the spleen. Further characterization of the TcdA response led to identification of a major peptide of interest that will aid in future investigation of the adaptive immune response following C. difficile infection.

Supported by grants from NIH (R01 AI158830-01, R21 AI164385)
The emergence of SARS-CoV-2 lineages and associated antibody responses among asymptomatic individuals in a large university community
Authors: Merling Marlena Rae , Mahfooz Najmus , Robinson Richard T
Published: August 2023

SARS-CoV-2 (CoV2) infected, asymptomatic individuals are an important contributor to COVID transmission. CoV2-specific immunoglobulin (Ig)—as generated by the immune system following natural infection or vaccination—has helped limit CoV2 transmission from asymptomatic individuals to more susceptible populations. The emergence of new Omicron lineages with extensive Spike and Nucleocapsid (N) mutations threatens our reliance on natural and vaccine-initiated Ig responses to limit COVID transmission; therefore, it is important to understand the relationships between CoV2 lineage emergence, CoV2-specific Ig levels, and their cross-neutralization capacity in asymptomatic individuals. Here, we describe the relationships between COVID incidence and CoV2 viral load, lineage, and CoV2-specific Ig levels (IgM, IgA, and IgG) in the saliva of asymptomatic individuals between Jan 2021 and May 2022. These data were generated as part of a large university COVID monitoring program and demonstrate that COVID incidence among asymptomatic individuals occurred in waves which mirrored those in surrounding regions, with saliva CoV2 viral loads becoming progressively higher until vaccine requirements were established. Among the unvaccinated, infection with each CoV2 lineage (pre-Omicron) resulted in Spike-specific IgM, IgA, and IgG responses, with Spike-specific IgG increasing significantly post-infection and being more pronounced than N-specific IgG responses. Among the vaccinated, saliva from those with breakthrough Delta infections had Spike-specific IgG levels that were comparable to those who were uninfected; their capacity to inhibit Spike function, however, was diminished relative to those who were uninfected.

The Ohio State University
Tissue engineering human thymus from iPSCs to support human T cell development
Authors: Fan Yong , McCormick Catherine , Liu Wen , Zeleniak Ann , Wiegand Connor , Krishnamurthy Ravi , Guan Haonan , Montanari Kaylee , Muraskin Nickia , Banerjee Ipsita
Published: August 2023

Thymus is the primary immune organ responsible for the generation of diverse and self-tolerant T cells that are essential for fighting off invading pathogens while maintaining immune homeostasis. Loss of thymus function, due to either genetic mutations or environmental insults, can lead to prolonged immunodeficiency that can be life-threatening. Rejuvenating the function of the thymus is challenging as the organ undergoes irreversible involution at young age, during which thymic epithelial cells (TECs) – the thymic stromal cells key to the education and selection of developing T cells – are progressively replaced by infiltrating adipocytes. Regeneration of thymus from the patient’s own stem cells will have tremendous clinical benefits in treating immunodeficiency and immunodysregulatory disorders.

We reported here the tissue-engineering of functional artificial thymus from inducible pluripotent stem cells (iPSCs). Taking advantage of the 3D alginate encapsulation technology, we developed a novel protocol to induce the differentiation and maturation of human iPSCs into TECs (iPSC-TECs) at high efficiency. To bioengineer the thymus, iPSC-TECs were injected into decellularized murine thymus scaffolds, in conjunction with T cell progenitors derived from hematopoietic stem/progenitor cells (HSPCs) harvested from the umbilical cord blood. When transplanted under the kidney capsules of hematopoietic humanized mice (aka immunodeficient mice engrafted with HSPCs), iPSC-TEC thymus can support developed a diverse population of human T cells that could mediate adaptive immune responses. Our findings suggest that functional human thymus can be generated from patient-derived iPSCs for regenerative medicine.

Supported in part by the NIH (R01 AI123392 and R21 AI126335), NSF (CBET 1804728), and Highmark Health Award (A023948-HIGHMARK-LANNI-FAN).
Chemotherapy-Induced Thymic Involution: An Ultrastructural Study
Authors: Lagou Maria K , Nishku Xheni , Churaman Joseph , Cummins Leslie , Macaluso Frank P , Karagiannis George S
Published: August 2023

Endogenous thymic repair is quite complex, demanding coordinated thymocyte and thymic stromal responses for T cell development and establishment of central tolerance. While the importance of thymic stroma, notably cortical (cTEC) and medullary (mTEC) thymic epithelial cells, has been shown in gain- and loss-of-function studies, endogenous thymic epithelial repair has never been resolved ultrastructurally. Using Transmission Electron Microscopy (TEM), we analyzed morphometrically thymi in mice receiving Cyclophosphamide (CTX). In vehicle-treated mice, the cortex was populous with thymocytes neighboring cTEC, which extended long cytoplasmic processes into a dendritic meshwork, called “cytoreticulum”. In CTX-treated mice though, thymocytes were scarcely met in the cortex and TEC appeared more circular/ellipsoid. The cytoreticulum was evidently collapsed, increasing total contact surface area among cTEC/mTEC subsets. Despite that thymic macrophages primarily mediate clearance of thymocytes failing positive/negative selection, those in CTX-treated thymi were rich in secondary lysosomes and many were found phagocytosing TEC. The surviving TEC in CTX-treated mice had increased autophagolysosomes per surface area unit of TEC cytoplasm compared to vehicle-treated ones. Analysis via intensity thresholding revealed higher electron density of enclosed particles, consistent with the presence of partly-digested amorphous material, indicating membranous organelle self-digestion (i.e., stress macroautophagy). These data propose an emerging hypothesis that survival of thymic epithelium following cytotoxic insult is mediated via a collective stress response involving macroautophagy activation in TEC.

Funded by: 1)AAI careers in immunology Fellowship - trainee 2)new investigator’s start up funds (PI)
Age-associated changes to lymph node architecture shape T cell responses
Authors: Lancaster Jessica N , Da Silva Ildefonso Alves , Kwok Tina , Fisher Jacob S
Published: August 2023

Aging dramatically impacts T cells, contributing to diminished and delayed responses to infection. While the development and maintenance of lymphocytes certainly undergo intrinsic changes with age, the contribution of cell-extrinsic forces, such as the aging tissue microenvironment, has been difficult to uncouple. Macroscopic changes to the lymph nodes have been well described, but the impact of aging on lymph node stromal subsets is less understood. Here, we visualized naïve T cell migration in the aged murine lymph node using live-cell 2-photon microscopy and determined that T cell motility was impaired in regions of increased fibrotic deposition. Using fluorescent reporters for Ccl19 and Cxcl12, homeostatic chemokines important for stromal:lymphocyte crosstalk, we also found that the distribution of stromal cells expressing these chemokines was altered with age, and that migrating naïve T cells were excluded from regions of chemokine expression. Flow cytometric analyses of fibroblastic reticular cells (FRCs) revealed decreased proportions of FRCs within the mesenteric lymph nodes and decreased putative FRC progenitors in the skin-draining lymph nodes. FRC progenitors at both sites had reduced surface expression of lymphotoxin-beta receptor, an important receptor for FRC differentiation and maintenance. We hypothesize that age-associated changes to FRC maturation contribute to increased lymph node fibrosis and altered localization of homeostatic factors. Current studies have indicated that in vitro cultured lymph node fibroblasts are sensitive to TNF-alpha and respond by increasing expression of alpha-smooth muscle actin, suggesting a mechanism by which aging remodels the lymph node microenvironment.
Influence of SLAM/SAP signaling on γδ T cell TCR signal strength during thymic development
Authors: Hilton Brianna M , Savard Remi , Dienz Oliver , Boyson Jonathan E
Published: August 2023

Innate-like T cells are unusual T cells that are enriched in mucosal tissues, where they constitute a prominent source of pro-inflammatory cytokines like IL-17 and IFN-γ. During thymic development, mouse γδ T cells commit to either γδT1, γδT2, or γδT17 phenotypes through mechanisms that remain unclear. Recent observations from our laboratory have suggested a role for the SLAM/SAP signaling pathway in γδ T cell thymic developmental programming. Here, we investigated the influence of the SLAM/SAP signaling pathway on γδ TCR signal strength during thymic development. Using Nur77GFP expression as a readout of TCR signal strength, we observed significantly greater Nur77GFP expression in B6.Nur77GFP.SAP−/− thymic γδ T cells compared their B6.Nur77GFP counterparts, suggesting that SAP inhibited TCR signal strength. Consistent with these findings, we observed significantly greater Erk phosphorylation in CD3-stimulated B6.SAP−/− thymic γδ T cells. In vitro stimulation of thymic γδ T cells with anti-TCRδ and anti-SLAM family receptors revealed that SLAMF6 co-stimulation resulted in decreased Erk phosphorylation, but increased Nur77GFP expression. Surprisingly, the effects of SLAMF6 co-stimulation on γδ TCR signaling were SAP-independent. Together, these data suggest an inhibitory role for SAP in γδ TCR signaling during thymic developmental programming.
Neural crest-derived mesenchymal cells support thymic reconstitution after lethal irradiation
Authors: Tetteh Doris Narki , Hikosaka-Kuniishi Mari , Yamazaki Hidetoshi
Published: August 2023

The thymus is composed of thymocytes and stromal cells including thymic epithelial and mesenchymal cells. Thymic mesenchymal cells are originated from Neural Crest (NC) cells and the mesoderm. Although it is known that NC-derived (NCd) cells contribute to thymus organogenesis and thymus development, their specific role in T cell regeneration is unclear.

To evaluate the regenerative ability of NCd mesenchymal cells in the thymus, we used lethally irradiated mice to examine the effect of ionizing irradiation (IR) on the thymus size, thymocytes and the thymic stromal cells (thymic epithelial cells (TECs) and thymic mesenchymal cells). Additionally, we ablated NC-derived mesenchymal cells via in vitro diphtheria toxin (DT) administration in double transgenic mice (Wnt1-Cre/+; RosaYFP/DTR mice) that express NC-specific Cre and Cre-driven DT receptor (DTR) and transplanted the thymus depleted of NC-derived mesenchyme under the renal capsules of non-irradiated C57BL/6 (CD45.1) mice.

IR reduced the size of the thymus, the absolute numbers and percentages of thymocytes, primarily (CD4+ CD8+) T cells and TECs. Moreover, IR reduced the number of thymic mesenchymal cells but not that of NC-derived mesenchymal cells. NC-derived mesenchymal cells upregulated several hematopoietic factors such as Flt3l, Periostin, Scf, and Bmp4 after irradiation and supported thymic reconstitution.

Taken together, our findings indicate that NC-derived mesenchymal cells are not only radio-resistant, but also produce molecules that may support early T-cell development for thymic reconstitution after irradiation .
Toll-like receptor expression on young and old hematopoietic cell types
Authors: Lowe Sara Kristen , Elberfeld Lauren , Bhowmick Debajit , Ratliff Michelle
Published: August 2023

Hematopoiesis is an essential process that produces white and red blood cells within the body. As the body ages, we have observed defects in the hematopoietic process including an increase in the number of stem cells, skewing of white blood cells down the myeloid lineage, and a decrease in the production of new B and T cells. Since hematopoietic aging skews the production of professional immune cells, the elderly population has a disadvantage in recognizing and clearing infections compared to young individuals. Immune cells use a common bacterial and viral recognition system, which includes toll-like receptors (TLRs), to identify common pathogen-associated molecular patterns (PAMPs) that bacteria and viruses produce. Upon exposure, immune cells will activate to complete two functions: to direct myeloid differentiation for acute responses and to recognize and eliminate the pathogens via the TNFα or type 1 interferon (IFN-1) signaling pathway.

Deficiencies in aging hematopoiesis, IFN-1 expression, and the TLR responses associated with IFN-1 in immune cells have been described in both mice and humans. The expression of these TLRs and any age-associated changes in that expression, as well as downstream signaling, are not as well established. Our goal is to identify which hematopoietic cells express the four TLRs associated with IFN-1 (TLR-3, −4, −7, −9) and if aging leads to changes in receptor expression and/or downstream signaling in human immune cells. Using flow cytometry, we evaluated receptor expression and the downstream effects of TLR engagement. The data reveals age-associated changes in IFN-1-related innate immune signaling pathways.

Supported by grants from NIH (R00 AG055717)
TLR2/6 agonist treatment after total body irradiation in mice and nonhuman primates induces proliferation of hematopoietic progenitor cells
Authors: Brickey Willie J , Caudell David , Olson John , Huang Guannan , Li Sirui , Macintyre Andrew N , Yang Shuangshuang , Hsu Martin , Chao Nelson J , Cline J Mark , Ting Jenny P
Published: August 2023

Exposure to high doses of radiation, whether intentional or accidental, may cause injury or death. Therapeutics are urgently needed for mitigating injuries caused by radiation exposure. Previously, we identified a synthetic lipopeptide agonist of TLR2/6, namely fibroblast-stimulating lipopeptide (FSL-1), that prolonged survival of mice exposed to lethal ionizing radiation and mitigated hematopoietic acute radiation syndrome. Here, this agent was tested in several mouse models as well as in nonhuman primates given total body irradiation followed 24 hours later with a single subcutaneous injection of FSL-1. These studies revealed the mitigation of radiation-induced loss of platelets and red blood cells, as well as the promotion of bone marrow progenitor cell recovery. These beneficial hematopoietic outcomes were accompanied with a lack of toxicity, showcasing the safe, efficacious application of a single dose of FSL-1 for mitigating hematopoietic dysfunction resulting from radiation. By investigating the mechanism of action of FSL-1 in response to radiation, we confirmed that FSL-1 radio-mitigation activity was mediated through a TLR2/MyD88-dependent process. In FSL-1 treated irradiated mice, we found enhanced numbers of proliferating Ki67+ bone marrow cells. Finally, examination of cellular pathways in mice and nonhuman primate bone marrow cells indicated FSL-1 induced the activation of NF-kB and MAPK signaling. These data suggest that FSL-1 promotes hematopoietic progenitor cell proliferation through the activation of cell survival and activation pathways downstream of TLR2/6 stimulation, and thus may serve to mitigate the injurious consequences of radiation.

Supported by grants from NIH (AI067798) and DOD Congressionally Directed Medical Research Program grant W81XWH-15-1-0574 (JMC).