bluuebunny/crossref_metadata_2025 huggingface dataset:
Single-cell RNA sequence (scRNAseq) analysis of T regulatory cells in relapsing remitting multiple sclerosis (RRMS)
Authors: Wang Yan , Seyedsadr Maryamsadat , Markovic-Plese Silva
Published: January 2023

Patients with RRMS have decreased numbers and function of Tregs, which may play a causative role in the pathogenesis of the disease. Here, we used scRNAseq to define Tregs populations in peripheral blood of 3 RRMS patients and 3 matched healthy controls (HCs) and compare their transcriptomes and function. We clustered sorted CD4+ CD25+ CD127− Tregs into active (a) and resting (r)Tregs. Our initial scRNAseq data analysis of the active Tregs identified 324 up-regulated differentially expressed genes (DEGs) and 208 down-regulated genes in RRMS in comparison to HCs. Upregulated genes include major histocompatibility complex (MHC) class I (HLAA, HLAB) and class II (HLADRB5) and TRCA gene, supportive of their activated status; ICAM2, involved in the migration to the CNS; ID3, involved in maintaining Treg pool and suppressive function; LAIR2, an indicator for exhaustive Tregs; NEAT1, whose silencing promotes Treg/Th17 balance, and TCF7, that limits Treg thymic generation. Genes with decreased expression include CD7, required for Treg homeostasis; costimulatory CD81; CTLA4 and JUNB required for optimal Treg function; IFTM1 and IF16 that may regulate IFN type I induction of Tregs; CXCR4; LGALS1 and LGALS3 coding for galectin 1 and 3 which inhibit Treg function; and NFKBIA and TNFAIP3, all suggestive of decreased Treg function in RRMS. Pathway enrichment analysis for DEGs in RRMS Tregs identified the strongest enrichment for type I IFN signaling pathway. Understanding the transcriptional changes involved in deficient Tregs functions in RRMS is crucial for better understanding of the disease pathogenesis and for a development of effective treatment for this disabling disease.

State of Pennsylvania grant: Deficient T regulatory Cell (Treg) Function in Relapsing Remitting Multiple Sclerosis (RRMS)
Spatial transcriptomics and single cell sequencing identifies a unique injury associated resident macrophage subpopulation and reveals differential response to injury
Authors: Erman Elise , Cheung Matthew D , La Fontaine Jennifer , Yang Zhengqin , Agarwal Anupam , George James
Published: January 2023

Acute kidney injury (AKI) is a growing concern in the U.S. and globally. Despite recovery of kidney function, patients remain at increased risk for developing chronic kidney disease (CKD). The cause of this increased risk is unknown and biomarkers to determine kidney injury are severely lacking. A potential candidate is the network of kidney resident macrophages interwoven throughout the tissue, and specifically a subpopulation of kidney injury associated macrophages (KIA) we first identified to appear after AKI and lack MHC II expression. KIA cells were localized within the tissue using Visium Spatial Transcriptomics and flow cytometry. Comparison of KIA cell kinetics between AKI and CKD indicated a correlation between the presence of KIA cells and the persistence of injury. Additionally, KIA cells have localized to the sight of greatest injury following an ischemic event. As kidney function recovers, KIA cells decrease in number and return to quiescent levels by 14 days post injury. In contrast, in the severe CKD model, KIA cells persist for up to six weeks, composing approximately 20% of the total kidney resident macrophage population. A less severe version of CKD results in fewer KIA cells but similar persistence, indicating appearance of KIA cells directly correlates with injury severity. Targeting KIA cells to either enhance or negate their function is a possible therapeutic target to reduce the risk of developing CKD following AKI. As we have identified KIA cells in multiple models of kidney injury, in the future they should be investigated as a potential injury biomarker. We hypothesize that KIA cells are uniquely involved in the pathogenesis of injury and may be a key to an AKI to CKD transition.

Supported by grants from the American Heart Association: (AHA 827257)
P66 is a bacterial “don’t eat me signal” that mimics mammalian CD47 and facilitates immune evasion by Borrelia burgdorferi.
Authors: Tal Michal Caspi , Hansen Paige , Ramadoss Nitya , Volk Regan , Zaro Balyn , Weissman Irving L
Published: January 2023

Innate immunity, the first line of defense against pathogens, relies on efficient elimination of the invading agents by phagocytes. Thus in the co-evolution of host and pathogen, pathogens developed mechanisms to dampen and evade phagocytic clearance. Here, we report that bacterial pathogens can evade clearance by macrophages through molecular mimicry of a mammalian anti-phagocytic “don’t eat me” signal. Using a high affinity structural probe for human CD47, a dominant “don’t eat me” signal, we discovered a bacterial protein that mimics CD47’s structure on the surface of Borrelia burgdorferi (Bb), a bacterial spirochete that can establish infection in mammals including Lyme Disease (LD). Blockade of the mimic promotes clearance of the infection in vivo. We identified P66, a known virulence factor, as the bacterial mimic of CD47. Finally, we determined that patients who return to health following LD infection are more likely to generate antibodies to p66 compared to patients who do not. This study demonstrates molecular mimicry as a means used by Bb to inhibit macrophages and evade phagocytic clearance; this mechanism may have broad implications for understanding host-pathogen interactions and the development of therapeutic strategies to combat bacterial infection.

Research reported in this publication was supported by the Fairbairn family foundation; Bay Area Lyme Foundation; the Younger family foundation; the Robert J. Kleberg, Jr., and Helen C. Kleberg Foundation; the Virginia and D. K. Ludwig Fund for Cancer Research; AML grant R01CA086017; the PCBC from NIHLB U01HL099999; as well as grant U19AI109662. M.C.T. was supported by Stanford Immunology training grant 5T32AI007290, and the NIH NRSA 1 F32 AI124558-01 award. L.B.T.D. was supported by a Stanford Diversifying Academia Recruiting Excellence fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Adenosine receptor signaling regulates effector T cell activation through the limitation of aerobic glucose metabolism.
Authors: Campillo-Gimenez Laure , Li Michael , Molina Elsa , Ernst Peter B
Published: January 2023

Background

In a mouse model of colitis, we previously reported the protective role of the adenosine axis on effector T (Teff) cell responses and disease progression. Inflammatory reactions are paired with cell metabolic reprogramming from oxidative phosphorylation (OXPHOS) to aerobic glycolysis which is also a hallmark of T cell activation. Thus, we sought to evaluate the role of the adenosine 2A receptor (A2AR) signaling in the regulation of T cell metabolism.

Methods

Teff cells were reactivated in the presence or absence of A2AR agonists. Gene expression of mTOR, HIF-1α and cMyc (transcriptional factors of glycolysis-related genes) were quantified by qPCR. T cell metabolic phenotype was evaluated by transcriptomic profiling (Nanostring nCounter® Metabolic Pathway Panel), as well as real-time extracellular flux analysis, glucose uptake, and ATP production assays.

Results

mTOR, HIF-1α and cMyc gene expression increased as soon as 2hr post stimulation. At this time point, transcriptomic analysis revealed an A2AR agonist-induced downregulation of pathways related to T cell activation (i.e. mTOR, MAPK, PI3K, NFκB and cMyc pathways) and energy metabolism (i.e glycolysis, autophagy, fatty acid synthesis) whereas oxidative metabolism pathways (OXPHOS, fatty acid oxidation, glutamine metabolism) were upregulated (Pathway Score q-value <0.05). Functional assays showed a decreased de novo glucose uptake and glycolysis rate after A2AR agonist treatment while OXPHOS capacity and ATP production were not modulated.

Conclusion

A2AR signaling provokes a rewiring of the activated T cell metabolism towards an oxidative state making adenosine a potential T cell metabolic regulator that can be exploited in the treatment of IBD.

Supported by NIH R01AI079145
Circadian control of macrophages in the tumor microenvironment.
Authors: Clark Amelia M , Altman Brian J
Published: January 2023

Introduction

All leukocytes tested to date have functional circadian clocks, and nearly every arm of the immune response is subject to circadian regulation. Circadian clocks instruct the time-of-day-dependent, rhythmic expression of genes in a tissue- and cell-specific manner. In macrophages (mΦs), the circadian clock regulates several factors that are critical to executing effective immune responses. Tumor-associated mΦs are major contributors to immune suppression in the tumor microenvironment (TME). Evidence suggests that metabolically stressful factors in the TME such as acidic pH and nutrient limitation promote mΦ-mediated immune suppression, and recent data point to dysregulation of the circadian clock downstream of metabolic stress.

Methods

We study the effect of TME-associated metabolic stress on the circadian clock of mΦs in vitro by culturing bone marrow-derived mΦs in conditions mimicking acidic pH and nutrient limitations that have been observed in the TME. To study the impact of mΦ-intrinsic circadian rhythms on tumorigenesis in vivo, we use mice genetically engineered to have a myeloid cell-specific disruption of the circadian clock via deletion of the key clock protein BMAL1.

Results

Oscillation of core clock proteins is altered in mΦs subjected to TME-associated metabolic stress. Additionally, we observe increased tumor growth in mice co-injected with mΦs whose circadian clocks were disrupted compared to mice co-injected with mΦs whose circadian clocks were functional.

Conclusion

Our data suggests that stressful conditions associated with the TME can alter the mΦ circadian clock, and that a functional circadian clock in mΦs can suppress tumor growth in a syngeneic murine tumor model of pancreatic cancer.

This research has been supported by the following fellowships and grants: 2021-Current: Wilmot Predoctoral Cancer Research Fellowship, Wilmot Cancer Institute, University of Rochester Medical Center, Rochester, NY 2020-2021: NIH T32 Training Grant in Cellular, Biochemical & Molecular Sciences, University of Rochester Medical Center, Rochester, NY
Prenatal Rejecter NK Cells Enhance Allo-specific Th17/Tc17 Cell Responses via TGF-beta1, IL-6 and GM-CSF.
Authors: Kang Hee Kap , Ott Katherine , Turner Lucas , Redden Catherine , Langereis Morgan , Alhajjat Amir , Shaaban Aimen
Published: January 2023

In utero hematopoietic cellular transplantation (IUHCT) has the potential to treat congenital benign cellular disease without chemotherapy or radiation. Prenatal tolerance is essential in this regard. Previous studies involving prenatally created chimeras have shown that immunologically tolerant NK cells (friendly, fNK) from engrafter mice suppress allo Tc1 responses, while intolerant NK cells (hostile, hNK) from rejecter mice enhance allo Tc1 responses. Given that Th17 cells are also involved in allograft rejection, we sought to determine if rejecter hNK cells induce/expand allo-specific Th17/Tc17 cells.

We examined the helper potential of prenatally educated hNK cells in non-tolerant prenatal Balb/c --- > B6.Thy1.2 rejecter chimeras. Naïve Thy1.1+ responder T cells and allogenic target cells were adoptively transferred into rejecter or naïve hosts that had been depleted of endogenous T cells and fNK cells. We found increased Th17/Tc17 alloimmunity in the responder Thy1.1+T cell population in rejecters when compared to naïve hosts. Additionally, through ELISPOT, we confirmed increased IL-17A production by responder T cells when co-cultured with allo-specific hNK cells from rejecter mice. Hostile NK cells from rejecter mice produce higher levels of Th17 driving cytokines, TGF-beta 1, IL-6 and GM-CSF when compared to naïve hNK.

From these results, we conclude that prenatal rejecter hNK cells have an enhanced helper effect on Th17/Tc17 alloimmunity in vivo and in vitro. These findings support an evolving paradigm that prenatally educated NK cell can regulate the balance between Th1/Th17 and Treg functions.

Supported by NIH R01HL103745 Lurie Children’s Hospital Research Foundation (to A.F.S.)
GPR65, a critical regulator of Th17 pathogenicity, is regulated by the CREB/CRTC2 pathway
Authors: Hernandez Jeniffer B
Published: January 2023

GPR65 has been shown to be a critical regulator of Th17 pathogenicity. Loss of GPR65 in mice results in a decrease in Th17 cells and reduced susceptibility to a mouse model of multiple sclerosis. The CREB/CRTC2 pathway has emerged as an important regulator of immune function. We have previously shown that the CREB/CRTC2 pathway modulates autoimmune disease by promoting differentiation of Th17 cells. In this study we performed RNA-seq to identify Th17 genes regulated by the CREB/CRTC2 pathway. Our RNA-seq analysis led us to uncover the first mechanism of regulation of the orphan receptor GPR65 by the CREB/CRTC2 pathway. We show that GPR65 is a target of the CREB/CRTC2 pathway through expression studies and chromatin immunoprecipitation. In addition, we show that targeting of GPR65 with small molecules alters the expression of IL-17A. Understanding the regulation of GPR65 will be crucial in developing small molecules to treat patients with Th17-mediated diseases.
ITK tunes the Th17/Treg switch response by controlling calcium dependent signaling.
Authors: Anannya Orchi , August Avery
Published: January 2023

Naïve CD4+ T cells can differentiate into pro inflammatory T helper type 17 (Th17) and anti inflammatory T regulatory (Treg) lineages following T cell receptor (TCR) activation. The strength of the TCR signal is tuned by Interleukin-2 inducible T cell Kinase (ITK) and ITK can tune the development of Th17 and Treg cells. ITK signals can also tune a Th17/Treg switch under Th17 conditions. How ITK mediated tuning of TCR signal control naïve CD4+ T cell commitment into Th17/Treg lineages is unclear. Here we used ITK inhibitors, conventional ITK and allele specific ITK (ITKas) IL17 GFP/Foxp3 RFP reporter mice, to track naïve CD4+ T cell commitment into Th17/Treg lineages. Under Th17 conditions, reduced TCR signal strength in absence of ITK activity/expression, tuned naïve CD4+ T cell fate by reducing Th17 differentiation and inducing switch to cells resembling Tregs expressing Foxp3. The ITKas model showed this switch is due to specific ITK inhibition. The switched Treg like cells resemble Tregs by expression of Treg markers (CD25, CTLA4, PD1) and suppression of effector T cell proliferation. Transcriptomic analysis of switched Treg like cells by RNA Seq indicate they resemble Tregs, with reduced Th17 (Rorc, Il17) and increased Treg (Foxp3, Nrp1, Ikzf2) gene expression. Analysis of chromatin accessibility by ATAC Seq indicated closed loci for Th17 (Rorc, Il17) genes. Activating intracellular calcium dependent pathways led to a loss of the Th17/Treg switch response in naïve CD4+ T cell commitment, effectively bypassing ITK mediated tuning of the TCR signal critical for the switch response. In conclusion, we show here ITK tunes a Th17/Treg switch response in naïve CD4+ T cell commitment under Th17 conditions by controlling calcium dependent signaling.

Supported by grants from NIH (NIH AI129422).
Splenic T-bet+ B cells exhibit stem-like features and constitutively generate antibody-secreting cells
Authors: Knox James J , Rosenthal Rebecca L , Johnson John L , Zhu Jinfang (Jeff) , Cancro Michael P
Published: January 2023

T-bet-expressing B cells (a.k.a. Age-Associated B cells or atypical B cells) directly contribute to antiviral and autoimmune responses and thus represent a promising target for immunomodulatory therapies. T-bet+ memory B cells (MBCs) form and persist long term in the spleen as a resident subset; however, the functional role of this tissue-based population and mechanisms for its maintenance are poorly understood. Here, we use T-bet reporter and fate mapping mouse models to investigate the homeostasis and fate of T-bet+ B cell subsets. We identified an elevated turnover rate of T-bet+ MBCs, with ~1.5% of the population labeling with BrdU per day and 5–10% of T-bet+ MBCs in cell cycle (G1 or S) at any given time. T-bet+ MBCs also displayed resistance to sub-lethal irradiation. Fate mapping and adoptive transfer studies demonstrate that splenic T-bethigh B cells (Tbet++ CD11c+) spontaneously give rise to T-betlow (Tbet+ CD11c−) and ultimately a variety of effector B cell subsets, including GCB-like cells, plasma cells, and T-bet− memory-like B cells, absent exogenous challenge or stimulation. T-bet+ MBCs preferentially give rise to short-lived (CD138+ B220+) versus long-lived (CD138+ B220−) bone marrow and splenic plasma cells, and adoptively transferred T-bet+ MBCs are sufficient to generate plasma IgG2c titers in IgG2a-haptolype naïve recipients. Considering their long-term maintenance in the spleen, these findings suggest T-bet+ MBCs represent a self-renewing, stem-like pool that contribute to the maintenance of antibody titers by constitutive differentiation into antibody-secreting cells.
The circadian clock gene ARNTL regulates the expression of CCR6 and Th17 related genes in human cells
Authors: Singh Satya , Parween Farhat , Edara Nithin , Zhang Hongwei , Gardina Paul , Otaizo-Carrasquero Francisco , Myers Timothy , Farber Joshua M
Published: January 2023

Previously we have shown that CCR6 expression uniquely defines a major branch of the Th effector/memory population that includes cells with both Th17 and Th17-associated phenotypes, therefore, studying the regulation of CCR6 will reveal molecular mechanisms important for controlling broad aspects of the functions of Th17 and associated cell types. Microarray analysis of CD4+ memory T cells from adult blood, based on CCR6 expression, revealed that among many transcription factors which were differentially expressed in the CCR6+ vs CCR6− cells, 8 were identified as regulators of circadian rhythm including ARNTL/BMAL1, a basic-helix-loop-helix-PAS domain transcription factor that as part of a heterodimer with CLOCK/NPAS1 is a central component of the circadian clock within the brain, and functions in analogous transcriptional circuits in peripheral tissues, including cells of the immune system. We found that ARNTL was progressively up-regulated among the CCR6-expressing cells and that it has a predicted binding sites in the promoter region of CCR6. Knocking down ARNTL using siRNA in CD4+ CCR6+ cells from adult blood or using Arntl knockout mice have demonstrated a role for this factor in regulating CCR6 as well as other Th-17 cell associated genes. Studying ARNTL and other clock proteins in Th17 cells opens a new line of investigation into relationships between the molecular control of circadian rhythms and the regulation of adaptive immunity, which may have implications for immune homeostasis and Th17-cell associated disease.

Supported by Intramural Research Program, NIAID, NIH.
Exploring the functionality of CD56+ MAIT cell populations across human tissues
Authors: Kammann Tobias , Parrot Tiphaine , Boulouis Caroline , Buggert Marcus , Leeansyah Edwin , Sandberg Johan K
Published: January 2023

Mucosa-associated invariant T (MAIT) cells are an evolutionarily conserved population of T cells serving a sentinel function at mucosal barrier sites to mediate immune protection. MAIT cells utilize their semi-invariant T cell receptor (TCR) to react to non-peptidic antigens derived from microbial riboflavin metabolites, presented on monomorphic MHC-class I-like MR1 molecules. In addition to TCR-dependent recognition of vitamin B2metabolites, MAIT cells sense and respond to local inflammatory cytokines in an innate-like manner. Surface expression of CD56 was previously shown to be associated with enhanced MAIT cell sensitivity to TCR-independent activation. However, the regulation, frequency, or relevance of CD56 expression on human MAIT cells is currently not well understood. Flow cytometry revealed heterogeneity of CD56 expression across donor-matched tissues obtained from human organ donors. Strikingly, the majority of liver MAIT cells expressed CD56, whereas expression was consistently lower in other tissues. We evaluated de novo CD56 expression on blood-derived sorted CD56-negative MAIT cells following IL-7 or antigen exposure which is likely to occur in the hepatic acute phase response. CD56-negative MAIT cells strongly upregulated CD56 expression after IL-7 stimulation and maintained CD56 expression for up to 19 days. Exposure to MR1-presented antigen induced CD56 expression similarly but required prior cell proliferation. In summary, we identify heterogeneity between MAIT cell populations of different tissue origin and determine the dynamic regulation of the CD56-associated innate-like characteristics to shed light on unknown aspects of MAIT cell diversity and immunobiology.
The unusual peptidoglycan cell wall of Borrelia burgdorferi and it’s role in Lyme disease pathogenesis
Authors: McClune Mecaila E , Dressler Julianne , Davis Mari , Jutras Brandon
Published: January 2023

Borrelia burgdorferi — the Lyme disease agent — does not produce typical bacterial proinflammatory compounds, so the genesis of Lyme arthritis (LA) is largely unknown. To better understand this manifestation, we’ve begun investigating B. burgdorferi peptidoglycan (PG). PG is a large polymer that protects bacteria from various stressors. While most double-membraned bacteria recycle PG fragments during cell wall turnover, B. burgdorferi lack this pathway, instead releasing ~45% of their total PG into their host per generation. Released PG can interact with the immune system as a pathogen associated molecular pattern (PAMP). To that end, we discovered PG lingers in the synovial fluid of LA patients and PG, alone, can cause arthritis in a mouse model. Virtually all bacteria produce PG, but the chemical composition of B. burgdorferi PG is unlike any previously described. We hypothesize the unique chemical structure of B. burgdorferi PG, coupled with shedding, impact host immune responses, and contribute to cell wall half-life and LA pathogenesis. To study this relationship, we developed an in vivo imaging system to track the real-time biodistribution of B. burgdorferi PG in mice. Our results suggest certain organs act as reservoirs, retaining material well after arthritis resolution. Apart from half-life, transcriptomic studies reveal that B. burgdorferi PG induces unique immunological responses in human PBMCs, that appear to be dictated by PG chemistry. Our findings provide a fundamental basis for understanding the complex interplay between a unique PAMP and a host, in addition to deciphering the role of the B. burgdorferi cell wall in Lyme disease pathogenesis, results that may lead to new therapeutic approaches to alleviate LA symptoms.

Supported by grants from NIH (R21 AI159800), USDA (VA-160113), the Steven & Alexandra Cohen Foundation, and the Bay Area Lyme Foundation
Basophil depletion alters host immunity, intestinal permeability and mammalian host-to-mosquito transmission in malaria.
Authors: Donnelly Erinn L , Céspedes Nora , Van de Water Judy A , Luckhart Shirley L
Published: January 2023

Malaria-induced bacteremia has been shown to result from intestinal mast cell (MC) activation (Chau et al. 2013, Potts et al. 2016, Céspedes et al. 2020). The appearance of MCs in the ileum and increased permeability to enteric bacteria are preceded by an early Th2-biased host immune response to infection, characterized by the appearance of IL-4, IL-10, mast cell protease (Mcpt)1 and Mcpt4, and an increase in circulating basophils and eosinophils. Given the functional similarities of basophils and MCs in the context of allergic inflammation and the capacity of basophils to produce IL-4 in large amounts, we sought to define the role of basophils in the development of increased intestinal permeability and in MC influx and the development of bacteremia in the context of malaria. Upon infection with non-lethal Plasmodium yoelii yoelii 17XNL, mice that lack basophils exhibited increased intestinal permeability and increased ileal MC numbers, without any increase in bacterial 16S ribosomal DNA copy numbers in the blood, relative to non-depleted mice. Analysis of cytokines, chemokines and MC-associated factors in the ileum revealed significantly increased TNFα and IL-13 at day 6 post infection (PI) in basophil-depleted mice compared to non-depleted mice. Moreover, network analysis revealed profound differences between depleted and non-depleted mice over time following infection in both systemic and local immune responses to parasites and translocated bacteria. Finally, basophil depletion was associated with significantly increased gametocytemia and parasite transmission to mosquitoes, suggesting that basophils play a previously undescribed role in controlling gametocytemia and in turn, mammalian host-to-mosquito transmission.

This work was supported by NIH NIAID RO1 AI131609 to Shirley Luckhart.
PD-1 restrains B cell dependent CD4+ T cell responses that promote susceptibility to Brucella infection
Authors: Dadelahi Alexis S , Ateya Mostafa Farghal Namish , Ponzilacqua-Silva Bárbara , Chambers Catherine A , Lacey Carolyn A , Moley Charles R
Published: January 2023

Brucellosis, caused by intracellular pathogens of the Brucella genus, often results in chronic and/or lifelong infection. While T cells can contribute to control of Brucella, this typically does not result in sterile immunity, and the mechanisms underlying the inability of T cells to clear infection remain unclear. We previously found that B cells promote susceptibility to Brucella in a CD4+ T cell dependent manner. Here we report that B cell MHCII expression enhances susceptibility to infection, and investigated the role of B cell antigen specificity and presentation in this process. We found that B cell receptor specificity facilitates enhanced susceptibility to infection as well as Brucella entry into B cells in vivo. Investigation of the role of individual B cell subsets demonstrated that follicular B cells can inhibit protective CD4+ T cell responses. Evaluation of CD4+ T cell populations revealed that B lymphocytes promote FoxP3 expression preferentially amongst T follicular cell populations following Brucella infection. Additionally, CD40L blockade enhances protection against Brucella in a B cell dependent manner concomitant with a decrease in T follicular regulatory cells (TFR). Interestingly, PD-1 blockade resulted in an increase in TFR populations as well as an increased susceptibility to Brucella infection in a B and CD4+ T cell-dependent manner. Together, these data suggest B and CD4+ T cell interactions within the follicle promote TFR cell populations which in turn could promote susceptibility to Brucella infection.

Supported by NIH Grant:1R21AI135160 NIH Grant:1R01AI150797
Cellular dynamics of immune evasion during Leishmania major infection
Authors: Zayats Romaniya , Mou Zhirong , Yazdanpanah Atta , Koh Wan Hon , Lopez Paul , Uzonna Jude E , Murooka Thomas
Published: January 2023

Despite the generation of a strong T cell response, clearance of Leishmania major is incomplete. Understanding of the persistence mechanisms is lacking, but Leishmania major driven induction of the immunosuppressive microenvironment through recruitment of regulatory T cells has been proposed to prevent parasite clearance in vivo. In the presented study, we used a novel TCR transgenic mouse model, where CD4+ T cells recognize an immunodominant peptide derived from Leishmania-glycosomal phosphoenolpyruvate carboxykinase (PEPCK), to visualize the dynamics of anti-L. major CD4+ T cell responses and to characterize mechanisms which restrain their effector function. We show that macrophage:T cell interaction dynamics were prolonged upon antigen recognition. This leads to a production of high levels of IFNγ and can be significantly suppressed by PEPCK-specific Tregs in vitro, as compared to polyclonal Treg controls. Treg antigen activation leads to a substantial increase in IL-10 levels, while decreasing IL-12, TNF, and IL-2 production. Intravital microscopy studies characterizing PEPCK-specific CD4+ T cell migration within skin lesions in mice show a significant recruitment of adoptively transferred effector T cells to the lesion site in vivo, displaying behaviours consistent with antigen recognition at early stages, but not at chronic stages. Imaging of infected FoxP3-GFP reporter mice show recruitment of Tregs to the lesion site, where they localize in the lesion, unlike the effectors cells which surround it. Collectively, our findings show for the first time that Leishmania-specific Tregs influence effector CD4+ T cell responses and this could be a mechanism that derives antigen persistence in L. major infection.

Research funded by a grant from the GSK-CIHR Partnered Chair Program
Loss of Paneth cells alters intestinal innate lymphoid cells and enhances weight gain in mice.
Authors: Joldrichsen Marisa R , Kim Eunsoo , Steiner Haley E , Cormet-Boyaka Estelle , Boyaka Prosper N
Published: January 2023

Paneth cells are a subset of small intestinal epithelial cells specialized in the production of antimicrobial products and cytokines. Gastrointestinal dysbiosis has been linked to many health concerns and is believed to be a contributing factor in obesity. Despite the role of Paneth cells as a major source of antimicrobial products, the contribution of these cells to health or disease conditions has not been fully explored. We examine the impact loss of Paneth cells would have on the gut microbiota and whether the subsequent dysbiosis could affect metabolic functions of the host and fat accumulation in a mouse model of diet induced obesity. For this purpose, were used wild-type and Sox9ΔIEC mice, which lack Paneth cells due to a Sox9 gene deletion within the intestinal epithelium. Compared to control wild-type mice, the small intestine of Sox9ΔIEC exhibited an altered profile of ILCs characterized by increase ILC2 and decrease ILC3 numbers. After exposure to a high-fat diet for 13 weeks, the Sox9ΔIEC mice gained more weight and had higher glucose intolerance. The intestinal homeostasis was also affected, with an increase in intestinal permeability and an increase in ILC1s within the small intestine of Sox9ΔIEC mice. In the abdominal fat Sox9ΔIEC mice showed increased numbers of immune cells including inflammatory macrophages, T cells, and B cells. Finally, fecal material transplantation experiments revealed that fecal material from the Sox9ΔIEC mice transfer the phenotype in recipient germ-free mice as indicated by a trend towards an increase in weight gain and immune cell infiltration of abdominal fat. These results highlight the importance of Paneth cells as regulators of metabolic functions of the host and fat accumulation.

Supported by grants from OSU and NIH.
Expanding the use of clustering and dimensionality reduction in high parameter flow cytometry data through machine learning for novel samples.
Authors: Lownik Joseph Cornelius , Mahov Simeon , Alkan Serhan , Merchant Akil , Kitahara Sumire
Published: January 2023

High parameter flow cytometry is a highly utilized tool for accurate immunophenotyping and diagnostics in immunology, oncology, and many other disciplines. Over the past decade, several methods have been developed for automated clustering and dimensionality reduction of high parameter flow cytometry data which has sped up and simplified the discovery of cell populations not observed by manual gating strategies. However, the input and output of such tools are stochastic in nature, thus making their results difficult to reuse with novel samples. To address this challenge, we present a method utilizing machine learning to predict cluster labels and dimensionality reduction coordinates on novel samples. For proof of principle, we utilized high parameter (22 marker) flow cytometry data which examines myeloid hematopoiesis on bone marrow aspirate samples. Training data for machine learning consisted of pooled, normal, bone marrow. Phenograph was used for initial population clustering and UMAP for dimensionality reduction. A random forest model was found to be most accurate in predicting Phenograph clusters (98%) on novel data while a k-nearest neighbors (knn) model was found most accurate for UMAP coordinate prediction. The utility of this model was observed by examining acute myeloid leukemia with aberrant immunophenotypes, which were not included in the training data set. For these samples, predicted clusters and UMAP coordinates correlated with early progenitor populations from the training set. This approach allows for a de novo sample clustering and dimensionality reduction assignment onto an already established and characterized model, allowing for rapid, automated interpretation of high dimensional flow cytometry data.

Supported by Cedars-Sinai Pathology and Laboratory Medicine Minigrant
Robust Serum- and Feeder-Free Expansion of Mouse B Cells In Vitro
Authors: Arora Hitesh , Babic Sandra , Lee Tracy , Rogers Trevor , Eaves Allen C , Louis Sharon A , Brown Mark A , Kokaji Andy I , Tabatabaei-Zavareh Nooshin
Published: January 2023

Mouse B cells are used in workflows to discover novel therapeutic antibodies or develop preclinical applications using genetically engineered B cells. In vitro expansion of B cells offers the opportunity to improve the recovery of antibody sequences and aids in the potential increase in the diversity of the B cell repertoire. Achieving robust expansion of B cells in culture can be challenging, as it typically requires the addition of serum, feeder cells, or specialized culture plates. The addition of serum can introduce unknown factors and result in lot-to-lot variability. Therefore, we have developed a first-to-market culture system that does not require the use of serum, feeder cells, or specialized culture plates to achieve robust in vitro expansion of mouse pan-B cells. Pan-B cells isolated from mouse splenocytes by immunomagnetic enrichment were cultured in serum-free ImmunoCultTM-XF B Cell Base Medium supplemented with ImmunoCultTM-ACF Mouse B Cell Expansion Supplement for > 9 days, starting with seeding densities of 100,000 cells/well and passaging every 3 days. Although pronounced donor variability was observed in the rate of expansion of viable cells, mean ~13-fold, ~150-fold, and ~200-fold rates were observed after 3, 6, and 9 days in culture, respectively (n=6 donors; range 94- to 328-fold at day 9). The expanded B cells also upregulated expression of activation and differentiation markers such as CD86 (mean 74%; n=6 donors; range 34 – 90%), CD138 (mean 68%; n=6 donors; range 57 – 73%), and CD267 (mean 79%; n=6 donors; range 49 – 91%) at day 9. The expansion supplement should facilitate mouse B cell studies for antibody discovery and diverse basic and translational applications.
The Minimal Information about MHC Multimers (MIAMM)
Authors: Vita Randi J , Mody Apurva , Overton James A , Buus Soren , Haley Stephen T , Willis Richard A , Sette Alessandro , Mallajosyula Vamsee , Peters Bjoern , Altman John D
Published: January 2023

The Minimal Information about MHC Multimers (MIAMM, miamm.lji.org) is a recently established data standard to be applied to publications utilizing these reagents. Available at miamm.lji.org, we explain how to easily represent multimer reagents in a standardized format using ontology terminology. Additionally, we provide a free, publicly available Multimer Validation Tool. This tool helps users adopt this new data standard and was proven to be generally applicable to real life use cases by its validation of the data present in the NIH Tetramer Core Facility and the nearly 18,500 multimer assays in the Immune Epitope Database (IEDB). As the scientific public adopts MIAMM, the quality, reproducibility, and annotatability of MHC multimer reagent data in the scientific literature will be improved.

Funding: JDA, RAW, and DLL acknowledge support from the contract for the NIH Tetramer Facility (75N93020D00005) from the Yerkes National Primate Research Center (P51OD011132), and the Emory Center for AIDS Research (P30AI050409). RV, JAO, AM, and BP acknowledge support from National Institutes of Health grant R24 HG010032. RV, JAO, AM, BP, and AS acknowledge support from National Institutes of Health contract 75N93019C00001
A combination of genetic factors and dietary tryptophan shapes gut microbial dysbiosis in a lupus-prone mouse model
Authors: Ma Longhuan , Brown Josephine , Kanda Nathalie , Terrell Morgan , Ge Yong , Mohamadzadeh Mansour , Morel Laurence
Published: January 2023

Gut microbial dysbiosis, impaired intestinal barrier and altered tryptophan metabolism have been reported in lupus patients. Dietary tryptophan modulated disease activity and modified the composition of microbiota in triple congenic (TC), B6.Sle1.Sle2.Sle3, lupus-prone mice. However, whether this modification is facilitated by tryptophan acting directly on the microbiota, or indirectly through lupus genetic susceptibility remains unknown. To address this question, we transplanted fecal samples from TC and B6 control mice into germ-free or antibiotic-treated B6 mice that were fed with high or low tryptophan. Tryptophan enriched different species of bacteria from transferred TC and B6 microbiota. A bloom of Ruminococcus gnavus, a bacterium associated with human lupus nephritis, only emerged in the recipients of TC microbiota fed with high tryptophan. Further, recipient mice showed distinct fecal metabolite profiles based on dietary tryptophan and the TC or B6 origin of the microbiota. Interestingly, both genetics and tryptophan impaired gut barrier integrity in recipient mice. These results suggest that the dysbiotic lupus microbiota is shaped by a combination of dietary tryptophan and genetic background, the latter most likely through autoimmune inflammation. In particular, the exclusive expansion of the pathobiont Ruminococcus gnavus by high tryptophan from the microbiota of lupus-prone mouse demonstrate that the interplay of genetic and environmental factors can influence the pathogenesis of lupus through modifying gut microbiota and gut permeability.

This work was supported by a grant from the NIH R01 AI43313 to Laurence Morel.
Synergistic effect of CXCR5+CD8+ TAb-supp cell therapy and mTOR inhibition (but not calcineurin inhibition) in suppressing alloantibody following kidney transplant in mice
Authors: Han Jing Light , Zimmerer Jason , Zeng Qiang , Chaudhari Sachi , Breuer Christopher , Bumgardner Ginny L
Published: January 2023

CXCR5+ CD8+ TAb-supp cells inhibit alloantibody (alloAb) production, AMR, and prolong graft survival in mouse models. To determine if this cell therapy is compatible with standard immunosuppression (IS) strategies, we investigate the interaction of CD8+ TAb-supp adoptive cell therapy (ACT) and IS [mTOR inhibitors (mTORi) or CNi] in a mouse model of kidney transplant (KTx). CXCR5+ CD8+ T cell ACT to CCR5 KO recipients on day 1 after KTx (D1) significantly suppresses D14 alloAb titer (1259±114) versus untreated controls (5951±630; p=0.0001). Administration of mTORi significantly inhibits alloAb titer (495±77; p<0.0001), while CNi does not (6473±434; p=ns). ACT combined with mTORi significantly reduces alloAb titer compared to ACT alone (387±63 vs 1259±114; p=0.0007). Conversely, CNi treatment partially negates the beneficial effects of ACT on alloAb titer (2385±114; p=0.03). Similarly, ACT reduced the quantity of D14 splenic germinal center (GC; GL-7+ Fas+ B220+) B cells compared to untreated KO recipients (4037±515 vs 5815±655 cells/mm3; p=0.03). Addition of mTORi to ACT preserves ACT-mediated suppression of GC B cells (4498±940 cells/mm3) while CNi negates ACT-mediated GC B cell suppression (6130±834 cells/mm3; p=0.04). Analysis of persisting GFP+ CXCR5+ CD8+ T cells D14 after ACT shows that concurrent mTORi treatment is associated with comparable quantity (1671±139 cells/mm3; p=ns), while CNi is associated with reduced quantity (900±83 cells/mm3; p=0.01)of transferred GFP+ CXCR5+ CD8+ T cells detected in the spleen compared to ACT alone (1601±116 cells/mm3). ACT with CXCR5+ CD8+ TAb-supp cells is optimal when combined with mTORi, not CNi, to significantly inhibit alloAb production and reduce quantity of splenic GC B cells.

Supported by NIH R01 AI083456 (to GLB), T32 AI106704-07 and F32 AI161844 (to JLH), CA016058, UL1TR002733, the OSU Division of Transplant Surgery, and the OSU College of Medicine.
DNA-PK inhibition plus immune adjuvants promotes CD8 TIL infiltration, neoantigen presentation, and diversifies the tumor-reactive TCRβ repertoire in B16 melanoma
Authors: Christians Allison , Sanchez Amelia , Albert Gabriella , Bradeen Xander GordonRay , Geng Degui , Chen Jiangli , Gonzalez-Rivera Daniela , Davila Eduardo
Published: January 2023

Despite significant improvements in cancer immunotherapies, enhancing immunogenicity of non-responsive tumors warrants further investigation. A prior drug screen of ~3,000 compounds identified NU7441, a DNA PK inhibitor, as an effective compound that promotes immunogenicity of various melanoma lines in vitro. In this study, we hypothesized that in vivo combination therapy NU7441, STING-L, and CD40 agonist will enhance tumor immunogenicity resulting in both expansion and increased cytotoxic activity of CD8+TCRβ tumor-reactive TILs in B16 melanoma models. Results obtained by flow cytometry demonstrated that combination treatment 1) significantly increased the ratio of CD8/CD4 TILs, 2) expanded several tumor-reactive TCRβ clones, 3) increased granzyme B production and expression of 4-1BB and PD-1, 4) increased ratio of DC/MDSC infiltration, and 5) potentially identified a novel cytotoxic CD8+CD11c+GR-1+ population. Additionally, the expansion of tumor-reactive TCRs was attributed to DNA-PKi’s ability to both expand and diversify the number of neoantigen transcripts resulting in a broader neoantigen expression profile. RNA-seq identified 27 unique neoantigens as potential novel targets in melanoma immunotherapy. TILs isolated from B16 tumors were co-cultured with dendritic cells transfected with tandem-mini genes, each encoding ~10 neoantigens, and T cells were analyzed by flow cytometry to quantify the TCRβ repertoire and functional response to neoantigens. We demonstrate combination treatment with NU7441, STING-L and CD40 agonist enhance antitumor responses through increased myeloid cell infiltration and sensitization of tumor cells to T cell-mediated killing from an expanded CD8+TCRβ repertoire.

Supported by R01CA207913 NCI BX004935-01, VA, Merit Award P30CA046934, NCI Leukemia and Lymphoma Society Department of Defense Gates Grubstake University of Colorado Anschutz Medical Campus Cancer League of Colorado
Monocyte-directed targeting of VEGFA in the melanoma microenvironment
Authors: Bu Melissa Tian , Chandrasekhar Pallavi , Ding Lizhong , Hugo Willy
Published: January 2023

VEGFA plays a critical role in the immunosuppressive cancer response and related immunotherapy resistance. Combined with immune checkpoint inhibitors (ICI) such as those against CTLA-4 and PD-L1, anti-VEGFA agents have demonstrated early clinical success in metastatic melanoma and renal cell carcinomas. To overcome toxicity limitations, thereby opening a wider therapeutic window, VEGFA should be targeted near its production source. Recently, localized co-targeting of TGFβ, an immunosuppressive factor, near CD4+ T cells through bispecific “trap” antibodies have demonstrated robust pre-clinical success. Thus, we assert the superiority of a bispecific antibody guiding a “trap” for VEGFA to its cellular source over global anti-VEGFA agents combined with ICIs. We set out to identify the cell population responsible for secreting VEGFA in the melanoma microenvironment. Upon re-analysis of two existing, independent single cell RNA Sequencing datasets of patient-derived melanoma treated with ICIs, we identified myeloid populations as the main producers of VEGFA. We specifically found a classical monocyte population (CD14+ FCN1+ S100A8+) to express high levels of VEGFA. Of note, we discovered significantly higher VEGFA expression in monocytes from non-responders versus responders to ICIs. VEGFA-expressing monocytes are localized to the intratumoral environment, as our analysis of PBMC-derived monocytes lacked high VEGFA expression in comparison to those that were tumor-derived. Based on our findings, we propose the design of a bispecific antibody that simultaneously targets VEGFA and CD14 to leverage the synergistic pro-immune effects and toxicity diminishment that result from localized inhibition.

Supported by the NIH (R01 CA236910) and Melanoma Research Alliance Young Investigator Award to Willy Hugo.
T-cell phenotype varies in distinct tumor microenvironments and CD57+ TFH cells are associated with disease progression and inferior survival in follicular lymphoma
Authors: Yang Zhi-Zhang , Kim Hyo Jin , Wu Hongyan , Tang Xinyi , Krull Jordan , Mondello Patrizia , Villasboas Jose , Novak Anne , Ansell Stephen
Published: January 2023

The tumor microenvironment (TME) plays a crucial role in mediating the tumor immune response, thereby affecting patient outcomes in follicular lymphoma (FL). Using CyTOF, we analyzed a cohort of 82 FL patients with biopsy specimens collected before the first treatment regimen was administered. Our results showed that there was a difference in the T-cell phenotype in patients with various TMEs. Patients with TMEs rich in monocyte/macrophages/NK cells had more CD8+ cells and tended to have fewer TN, but more TEMRA and TEXH cells. Patients enriched with T cells exhibited a T-cell phenotype skewed toward to TCM and patients with B cell dominance tended to have significant higher number of TFH cells. T-cell phenotypes with more terminally differentiated, senescent and exhausted cells were highly represented in patients with less T cells when compared to patients with more T cells. We observed that CD57+ TFH cells was significantly more abundant in patients who had disease progression than patients who had complete response to therapy, suggesting a role for CD57+ TFH cells in promoting malignant cell growth in FL. Consistent with this finding, we observed that increased numbers of CD57+ TFH cells correlated with an inferior survival in FL. Using CITE-seq technology, we found that CD57+ TFH cells exhibited a substantially different transcriptome from CD57− TFH cells. Genes that were differentially upregulated in CD57+ TFH cells when compared to CD57− TFH cells included genes involved in cell survival, compromised inflammatory response, and metabolic activation. Taken together, our results indicate different tumor microenvironments among patient groups that is associated with variable T-cell phenotypes.