bluuebunny/crossref_metadata_2025 huggingface dataset:
Leveraging outbred mice to identify genetic predictors of durable response to anti-tumor antibody
Authors: Glassbrook James E , Hackett Justin , Muñiz Maria , Gibson Heather
Published: February 2023

Trastuzumab is one of many monoclonal antibody (mAb) therapies targeting tumor-associated antigens (TAA) that have markedly improved cancer outcomes. Trastuzumab targets epidermal growth factor 2 (HER2). While it has long been considered the standard-of-care for HER2+ breast cancer treatment, differential response between patients suggests that host genetic background may contribute to an effective response to therapy. We utilize genetically heterogeneous Diversity Outbred (DO) mice to identify genetic elements that influence host response to TAA-mAb therapy. In a cohort of (DOxBALB/c)F1 mice bearing established BALB/c-syngeneic Neu+ (a HER2 homolog) TUBO mammary tumors, we find a divergent response to αNeu mAb therapy (clone 7.16.4). Several phenotypes were collected to compare immunological function to projected tumor doubling time (TDT). These include activation of peripheral anti-tumor T cells and IgG, natural killer cell ADCC competence, bone marrow derived macrophage ADCP/trogocytosis efficacy, and tumor immune cell infiltrate proportions. Contrary to the prospective mechanism of action of trastuzumab, we show that neither capacity for ADCC or ADCP correlate with a robust response to therapy. Following genetic linkage analysis, we identify a locus on Chr 10 that associates with TDT. To verify this locus, we use recombinant inbred collaborative cross (CC) mouse strains (CCxBALB/c) F1 mice that contain or lack this driver genotype. We find pooled survivorship between prospective responder/non-responder strains 2 months post tumor implantation differs by ~8%. This locus alone can’t fully account for a durable response to mAb therapy highlighting the multigenic nature of host immune response.
Multicellular immune hubs and their organization in MMRd and MMRp colorectal cancer
Authors: Chen Jonathan H , Pelka Karin , Hofree Matan , Giannakis Marios , Boland Genevieve M , Aguirre Andrew J , Anderson Ana C , Rozenblatt-Rosen Orit , Regev Aviv , Hacohen Nir
Published: February 2023

Immune responses to cancer are highly variable, with mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. To understand the rules governing these varied responses, we transcriptionally profiled 371,223 cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd patients. Analysis of 88 cell subsets and their 204 associated gene expression programs revealed extensive transcriptional and spatial remodeling across tumors. To discover hubs of interacting malignant and immune cells, we identified expression programs in different cell types that co-varied across patient tumors and used spatial profiling to localize coordinated programs. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage, and an MMRd-enriched immune hub within the tumor, with activated T cells together with malignant and myeloid cells expressing T-cell-attracting chemokines. By identifying interacting cellular programs, we thus reveal the logic underlying spatially organized immune-malignant cell networks.
Targeted protein degradation via NEDD8 conjugation does not enhance direct MHC class I antigen presentation.
Authors: Vijayasimha Kartikeya , Leestemaker-Palmer Amy Leigh , Gibb James , Yewdell Jonathan W , Dolan Brian P
Published: February 2023

Knowledge of different pathways contributing to peptide generation for direct MHC class I antigen presentation is important to the field of immunotherapy. Here we investigated the role of the ubiquitin-like protein NEDD8 in antigen presentation. We show that proteins tagged N-terminally with NEDD8 undergo rapid degradation via the proteasomal, and autophagy-lysosomal pathways. To determine if protein NEDDylation increased peptide presentation, we fused NEDD8 to the N-terminus of a cytosolic form of ovalbumin (OVA) and measured presentation of the SIINFEKL peptide via the murine MHC class I molecule K b , and compared the results to ovalbumin N-terminally modified with ubiquitin. While NEDD8-OVA was processed for degradation via the proteasomal and autophagosomal pathway Ub-OVA was only degraded via the proteasome. Peptide presentation from NEDD8-OVA was dependent on NEDD8-ultimate buster 1 (NUB1), while autophagy did not contribute peptides antigen presentation. Overall, we found that N-terminal Ub conjugation was far more effective at generating antigenic peptides than N-terminal NEDD8 conjugation. However, upon inhibition of NEDDYylation by treatment with MLN4924, we did detect a decrease in peptide presentation from a model antigen used specifically to track Defective Ribosomal Product (DRiP) presentation. These data demonstrate that while NEDD8 can cause the rapid degradation of substrates it is not as efficient as Ubiquitin for generating antigenic peptides. Our data provides a basis for understanding the role of NEDD8 in protein degradation and antigen presentation, especially in conditions of NEDD8 dysregulation, such as in some cancers and protein folding pathologies.
Epithelial antigen presentation regulates CD4+ TRM cell locations, functions and activities
Authors: Shenoy Anukul T. , Arafa Emad I , De Ana Carolina Lyon , Barker Kimberly A , Korkmaz Filiz T , Ramanujan Aditya , Etesami Neelou S , Martin Ian MC , Tilton Brian R , Hinds Anne , Goltry Wesley N , Kathuria Hasmeena , Jones Matthew R , Quinton Lee J , Belkina Anna C , Mizgerd Joseph P
Published: February 2023

Barrier tissues are sentinelled by CD4+ TRM cells with potent anti-microbial activities and considerable lineage plasticity. We hypothesized that local antigen presentation by lung epithelial cells (LECs) instruct CD4+ TRM cell activities. Pneumococcal infections in transgenic mice, flow- and spectral-cytometry, computational biology, and immunofluorescence were used to study this biology. All LECs including a novel alveolar surfactant protein C (SPC)low LEC were adept at antigen presentation. Temporal analysis of LECs for MHC-II and costimulatory/coinhibitory molecules revealed that airway club cells were T-cell stimulatory via CD40 while alveolar LECs expressed T-cell inhibitory PD-L1. This anatomical segregation of LEC antigen presentation correlated with deposition of CD4+ TRM cells around airways such that ablation of LEC MHC-II disrupted CD4+ TRM niches and blockade of CD40 signals prevented accumulation of CD4+ TRM cells. Recurrent memory recalls in absence of LEC MHC-II led to expansion of unconventional CD4+ TRM cells co-expressing classically incompatible lineage-defining transcription factors, changing their cytokine repertoire and leading to dysregulated immunity that phenocopied clinical features of checkpoint blockade therapy. Consequently, a tight correlation between MHC-II and PD-L1 was confirmed in mouse and human LECs. We discovered that LEC MHC-II functions in post-translational trafficking lockstep with PD-L1 to exert its restraints on TRM cell activities. Our results identify epithelial antigen presentation as critical instructors of CD4+ TRM cell locations, phenotypes and activities and establish epithelial-CD4+ TRM cell immunological synapses as key components of barrier immunity.
Sex hormones play a critical role in modulating the immune response to peanut
Authors: Vininski McKenna S , Dolence Joseph J.
Published: February 2023

The prevalence of peanut (PN) allergy is increasing rapidly. While PN allergy is a major medical and economic problem, our knowledge of the immunological mechanisms involved in the development of the disease remains limited. Sexual dimorphisms have been observed in allergic asthma and in autoimmune diseases such as systemic lupus erythematosus. However, how sex hormones regulate the immune pathways that lead to the development of PN allergy is unknown. This study aims to better understand how sex hormones impact the development of allergic responses to PN. Male and female wild type C57Bl/6 mice were sensitized to PN in our established four-week PN inhalation mouse model. Wild type (WT) males had significantly less IgE levels than WT females. When WT male and female C57Bl/6 mice were gonadectomized and implanted with capsules containing opposite sex hormone (e.g. β-estradiol in males and dihydrotestosterone in females), data showed females treated with testosterone displayed reduced allergic reactions to PN. Taken together, this data strongly suggests that sexual differences exist in the development of PN allergy and furthermore, supports that testosterone plays a role in modulating allergic immune responses to PN.
IL-9 regulates type-2 CD4 tissue resident memory cell mediated allergic airway recall responses
Authors: Kharwadkar Rakshin P , Ulrich Benjamin J , Chu Michelle , Fu Yongyao , Pajulas Abigail , Kaplan Mark H
Published: February 2023

Allergic asthma is a chronic inflammatory lung disease with intermittent flares where patients experience symptoms mediated by memory T cells. Allergen-specific CD4+ tissue resistant memory (Trm) cells in peripheral tissues have been reported to facilitate mucosal barrier immunity. However, the precise role of Trm CD4+ T cells in allergic exacerbations is not clearly defined. In this study we identified interleukin-9 (IL-9)-secreting allergen-specific CD4 T cells with a memory phenotype that regulate recall response to Aspergillus fumigatus. Inhibition of circulating T cells by administering FTY720 in the last month of rest, diminished the total CD4 T cells population in the lung, while the IL-9 secreting CD4 tissue resident cells remained stable. Single-cell RNA-seq and ATAC-seq confirmed the presence of multi-cytokine producing IL-9 secreting CD4 Trms in early recall responses to the allergen. Blockade of IL-9 prior to recall challenge phase reduced overall allergic lung inflammation observed through both flow-cytometry and single cell-RNA-seq analysis. IL-9 neutralization reduced cellularity of several populations in the lung including granulocytes and lymphocytes. Moreover, the expression of genes associated with mucus metaplasia including Muc2, Muc5ac, Muc5b, and Bpifb1 were significantly reduced in type-2 alveolar and epithelial cells with anti-IL-9 treatment. These findings demonstrate that IL-9-producing Trms play an important role in mediating allergic memory responses. IL-9 could be a promising therapeutic target specifically in patients showing symptoms of intermittent allergic response.
Suppression of T-cell Immune responses by Neutrophils in a direct contact manner
Authors: Soyemi Olamipejo O. , Moreira Josimar Dornelas , Samten Buka
Published: February 2023

Neutrophils are protective against early Mycobacterium tuberculosis (Mtb) infection, but overactive neutrophil response is often associated with chronic lung inflammation, tissue damage and compromised T-cell immunity, implying potential immunosuppression by neutrophils in Mtb infection. To test this, we examined the effect of peripheral blood neutrophils on the immune responses of T-cells from the peripheral blood mononuclear cells (PBMC) of persons with latent tuberculosis infection. Co-cultures of PBMC with autologous neutrophils at different ratios inhibited Mtb-stimulated T-cell proliferation and cytokines IFN-γ, IL-17 and IL-10 production dose dependently without affecting cell viability as determined by resazurin assay for cell metabolism and annexin V/PI staining for cell death. Neutralization of IL-10 and TGF-β failed to rescue neutrophil suppression of T-cell IFN-γ production. Neutrophils suppressed IFN-γ mRNA expression as determined by qPCR. Neutrophils inhibited purified T-cells stimulated with immobilized anti-CD3 and anti-CD28 mAbs, suggesting direct effect on T-cells without the involvement of antigen presenting cells. Trans-well study showed that cell-cell contact is required for this suppression. Further assessment of granulocyte derived suppressor cell phenotype and blocking the interaction between immune checkpoint molecules, PD-1 and PD-L1, refuted their roles in suppression of T-cells responses in Mtb infection. Together, these data suggest that neutrophils suppress T-cell immune responses by direct cell-cell contact mechanism and identification of molecular details of such suppression will shed new insights into the pathogenesis of tuberculosis and other infectious diseases.
Immunosuppressive function of DPP4, a proinflammatory molecule, in inflammatory colitis
Authors: Zhong Jixin , Wei Yingying , Rao Xiaoquan
Published: February 2023

We and others have shown that dipeptidyl peptidase-4 (DPP4) promotes inflammation in a number of conditions. However, it’s role in inflammatory colitis is not fully understood. Here we examined the role of DPP4 in dextran sulfate sodium (DSS)-induced colitis model and T cell transfer model of chronic colitis. The expression of DPP4 was significantly decreased in the colon tissue of wild-type (WT) mice with colitis. The mortality and disease activity score were markedly increased in mice deficient for DPP4 (Dpp4−/−), compared to WT mice. Chimeric mice with Dpp4−/− bone marrow showed a similar disease severity to whole body DPP4 knockout mice in DSS induced colitis, suggesting that bone marrow-derived immune cells were responsible for the protective effect of DPP4 on colitis. Flow cytometry analysis indicated that DPP4 was mainly expressed in T cells and dendritic cells (DCs) in mesenteric lymph nodes. Using T cell transfer model of chronic colitis in Rag2−/− mice or Dpp4−/− Rag2−/− mice, we found that innate immune cells other than adaptive immune cells were responsible for the protective effect of DPP4. Subsequent flow cytometric analysis showed that DPP4 was mainly expressed on a DC subset that was responsible for the induction of regulatory T cells (Tregs) in the gut. RNA sequencing data also indicated that expression of genes in TGFβ signaling pathway was significantly reduced in Dpp4−/− mice, accompanied by an increased expression of multiple inflammatory genes. Co-transfer of WT Tregs protected Rag2−/− mice, but not Dpp4−/− Rag2−/− mice, from developing colitis after transfer of CD4+ naive T cells. Our data suggest that DPP4 expression on innate immune cells may be responsible for maintaining Treg’s suppressive function in colitis.
The Ubiquitin Ligase Itch Regulates Germinal Center Selection
Authors: Moser Emily K
Published: February 2023

More than 23 million Americans suffer from autoimmune diseases driven by somatically mutated autoantibodies. High levels of somatic hypermutation of immunoglobulin genes occurs in germinal center (GC) B cells. Some of these mutations render autoreactivity, but cells bearing autoreactive receptors are eliminated due to strict selection of GC B cells by mechanisms that are still poorly defined. Inappropriate positive selection signals can rescue autoreactive cells from elimination and drive autoimmune disease. The E3 ubiquitin ligase Itch prevents the emergence of autoimmune disease and autoantibodies in humans and mice, and patients lacking Itch develop debilitating, multi-faceted, potentially fatal, autoimmune disease; yet how Itch regulates GC B cell fate or function is not well understood. By studying Itch deficient mice, we have recently shown that Itch directly limits B cell activity to shape antibody responses. While Itch-deficient mice displayed normal numbers of pre-immune B cell populations, they showed elevated numbers of GC B cells. Mixed bone marrow chimeras revealed that Itch acts within B cells to skew the proportions of light zone and dark zone GC B cells. Proteomic profiling of GC B cells uncovered that Itch deficient cells exhibit hallmarks of positive selection. These results support a novel role for Itch in limiting positive selection of GC B cells to restrict GC size. In this role, Itch may shape the GC-derived antibody repertoire to prevent autoimmunity.
Lessons from humanized mice: Is Foxp3 a bona fide marker of human regulatory T cells?
Authors: Morina Lyra , Ward Kirstin , Shevach Ethan M , Buszko Maja
Published: February 2023

Regulatory T cells (Tregs) suppress conventional T cells (Tconv) and are critical for homeostasis of the immune system. Tregs are identified by the transcription factor Foxp3 and, to a large extent, by the transcription factor Helios (expressed by 80% of human Tregs). One confounding factor in the study of human Tregs is that Tconv cells upregulate Foxp3 expression upon activation in vitro. However, it remains unclear whether this is the case in vivo. To investigate differences in phenotype between Tregs and activated Tconv cells, peripheral blood mononuclear cells (PBMCs) from healthy human donors were activated in vitro and in vivo and analyzed for expression of activation markers and cytokines using flow cytometry. For in vitro activation, PBMCs were stimulated in xenogeneic reactions with mouse splenocytes or with anti-CD3. For in vivo activation, human PBMC-engrafted NOD-scid IL2Rgnull (NSG) mice were used. NSG mice develop xenogeneic graft-versus-host disease (GVHD) as human cells begin to attack mouse tissues. During in vitro activation, there was an increase in Foxp3 expression by IL-2 producing, Helios− Tconv cells. On the contrary, during the course of GVHD in vivo, activated Tconv cells, identified as CD25+ and Foxp3−, produced IL-2 but remained Foxp3− and Helios−. Foxp3+ Tregs did not produce IL-2 and remained predominantly Helios+. In conclusion, while expression of Foxp3 is not a reliable marker of activated human Tregs in vitro, it appears to be a specific marker of activated human Tregs in vivo as observed in this humanized mouse model. Using Foxp3 to distinguish human Tregs can improve diagnosis and treatment for autoimmune disorders and cancer.
Critical role for adhesion and degranulation promoting adaptor protein in homeostasis and function of invariant natural killer T cells
Authors: Das Rupali , Bahal Devika , Gohl Trevor , Lee Hyun Hee
Published: February 2023

Invariant natural killer T cells (iNKTs) are innate T lymphocytes that are critical in modulating immune response in cancer, infection and inflammation. However, the mechanisms that regulate iNKT cell development and function are not fully known. In the current study, we demonstrate that adhesion and degranulation promoting adaptor protein (ADAP) is dispensable for iNKT cell thymic development but is required for their peripheral maintenance. Strikingly, ADAP-deficient (Adap−/−) mice have significantly reduced iNKTs in the periphery that is associated with decreased surface expression of the critical homing receptor (CXCR6), homeostatic proliferation and increased apoptosis. Consistently, Adap−/− iNKTs have markedly reduced expression of pro-survival factors (PLZF, ICOS, GATA-3 and Id2) but increased levels of pro-apoptotic molecules (Bax, Bad and Bim). Functionally, Adap−/− iNKTs have significantly reduced cytokine production and bystander immune cell activation as well as poor anti-tumor response, both in vitro and in vivo. In agreement with these findings, ADAP-silenced human iNKTs have remarkably impaired cytokine production and cytotoxicity against leukemia targets in vitro. Mechanistically, preformed transcripts for interleukin (IL)-4 and IFN-g as well as several microRNAs are reduced in Adap−/− iNKTs. Additionally, these cells exhibit reduced capacity to form conjugates and have diminished actin polymerization at the immunological synapse. Collectively, our findings establish a novel and pivotal role for ADAP in iNKT cell homeostasis and function.
Vitamin C Potentiates Plasma Cell Differentiation via TET-mediated DNA Modifications at a Novel Prdm1 Distal Element
Authors: Chen Heng-Yi , Alamonte-Loya Ana , Lay Fang-Yun , Johnson Eric , Gonzalez-Avalos Edahi , Yin Jieyun , Wozniak Daniel J. , Ghoneim Hazem , Harrison Fiona , Lio Chan-Wang Jerry
Published: February 2023

Ascorbate (vitamin C) is an essential micronutrient in humans. Chronic severe ascorbate deficiency results in a condition termed Scurvy with increased susceptibility to infections. How ascorbate affects the immune system at the cellular and molecular levels remained unclear. In this study, we showed that ascorbate is pivotal for the IL-21-dependent differentiation of BLIMP1+ plasma cells. Ascorbate significantly increases the proportion of B cells expressing BLIMP1 upon IL-21 stimulation without affecting the IL-21-STAT3 signaling. As a co-factor for TET DNA 5-methylcytosine oxidases, ascorbate boosters the enzymatic activity of TET, promoting the oxidation of 5-methylcytosines into 5-hydroxymethylcytosine (5hmC), an intermediate for DNA demethylation. Correspondingly, TET2/3 are required for the enhancement of plasma cell differentiation by ascorbate. Using 5hmC enrichment analysis, we identified ascorbate promotes plasma cell lineage via a novel distal element at the BLIMP1/Prdm1 locus. This ascorbate-responsive element (EAR) is normally methylated and is hydroxymethylated in the presence of ascorbate. Notably, the 5hmC-modified CpG exists within the STAT3 binding motif and the pattern is evolutionarily conserved, suggesting the ascorbate-assisted DNA modification is required for STAT3 recruitment to EAR and the subsequent Prdm1 activation. This study provides an example of how micronutrients may influence immune response via epigenetic regulation. Our finding also implies that epigenetic enzymes can function as sensors to gauge the availability of metabolites and influence cell-fate decisions.
Tonic sympathetic nervous system signaling regulates the maturation of natural killer cells
Authors: Geyer Rory , Moussa Marmar , Mandoiu Ion I. , Srivastava Pramod K. , Nevin James T.
Published: February 2023

Natural killer (NK) cells play a critical role in immune responses to certain viral infections and cancers by directly killing infected or transformed cells and producing cytokines that may enhance the function of other immune cells. NK cell effector function depends on the acquisition of a variety of activating and inhibitory receptors on their surface. NK cells that have acquired the full complement of these surface receptors are considered “mature.” While maturation of NK cells is essential for their proper function, knowledge of specific signals that contribute to their maturation is lacking. Here, we find that one such signal comes from the sympathetic nervous system (SNS). Single-cell RNA-sequencing and flow cytometric characterizations of mice injected with the sympatholytic compound 6-hydroxydopamine (6-OHDA) reveal that they have reduced frequencies of mature CD27−CD11b+ NK cells and increased frequencies of their immature precursors, CD27+CD11b− and CD27+CD11b+ NK cells in the spleen and bone marrow. Similarly, NK cell recovery after depletion is altered by SNS ablation, with the NK cells that recover in 6-OHDA-treated mice being unable to reach full maturity. SNS ablation also leads to accelerated growth of RMA/S tumors, the rejection of which depends on NK cells. The data presented here, along with further characterization of the effects of SNS signaling on NK cells will enhance our understanding of how the nervous system and immune system communicate with each other and how this communication influences immune responses to infection and cancer.
Endoplasmic reticulum associated degradation adaptor Sel1L regulates T cell survival and homeostasis
Authors: Dils Alexander Thomas , Correa Luis O. , Gronevelt Jessica Paige , Liu Lu , Kadiyala Padma , Li Qing , Carty Shannon A.
Published: February 2023

Suppressor/Enhancer of Lin-12-like (Sel1L) acts as a critical adaptor in endoplasmic reticulum-associated degradation (ERAD), a process that maintains cellular protein quality control through the degradation of misfolded proteins. Here, we investigate the role of Sel1L in mature T cells. CD4Cre-driven deletion of Sel1L in murine models resulted in a slight decrease in the frequency and absolute number of CD4 and CD8 single-positive thymocytes with a profound reduction in peripheral T cells. Using mixed bone marrow chimeras, Sel1L regulates peripheral T cell survival in a cell-intrinsic manner. Sel1L-deficient T cells have increased AnnexinV staining suggesting Sel1L deletion promotes apoptosis. Furthermore, the Sel1L conditional knockout mice have a marked defect in the naïve CD8+ T cell compartment, which also occurs in a cell-intrinsic manner. These data suggest that Sel1L is critical for the maintenance of naïve CD8+ T cell homeostasis. Sel1L-deficient T cells exhibit enhanced mTORC1 activation with increased cellular phosphorylated S6 ribosomal protein and phosphorylated eukaryotic translation initiation factor 4E-binding protein 1 (4-EBP1), as well as altered cellular metabolism. These results identify a novel role for Sel1L in T cell survival and homeostasis.
Differential response of young and aged Virtual Memory CD8 T (TVM) cells to helminth-induced Th-2 cytokines
Authors: Hussain Tabinda , Nguyen Angela , Daunt Carmel , Thiele Daniel , Smith Tiffany , Harris Nicola , Quinn Kylie , La Gruta Nicole
Published: February 2023

TVM cells are antigen-naïve, semi-differentiated CD8 T cells that develop in response to common γ (γc) chain cytokines, like IL-15 and IL-4. TVM cells respond more rapidly than conventional naïve CD8 T cells to antigen stimulation, thereby providing early control of viral and bacterial infections. TVM cells comprise 20% of the naive CD8 T cell pool in young mice but are selectively retained and make up to 50% of all antigenically naïve CD8 T cells in aged mice. In addition, aged TVM cells exhibit markers of senescence and lose proliferative capacity compared to young TVM cells. Recently, it was shown that IL-4 generated during helminth infection drove TVM cell expansion and improved pathogen clearance in subsequent infections. However, it is not known if the expanded subset is maintained with age or whether helminth infections similarly expand or improve the function of aged TVM cells. To evaluate this, we infected young mice with the helminth, Heligmosomoides polygyrus, and aged them to over 18 months. There was no difference in number or function of TVM cells in previously infected as compared to naïve aged mice. We then infected aged mice with helminths and observed no increase in TVM cell numbers, but there was a reduction in IFN-γ production by aged TVM cells. When naïve TVM cells were transferred to a young helminth infected host, proliferation of aged TVM cells was markedly reduced as compared to young TVM cells. Collectively these data suggest that, the protective effect of helminth infection in young mice, mediated by TVM cell expansion, is firstly not maintained into advanced age. Secondly, this effect cannot be generated in aged mice due to aged TVM cells being intrinsically less responsive to the helminth-induced cytokine environment.
Adaptive γδ T cell function is directly subverted by the Yersinia pseudotuberculosis outer protein YopJ
Authors: Chu Timothy H , Khairallah Camille , Shieh Jason , Cho Rhea , Qiu Zhijuan , Zhang Yue , Jayatilaka Indralatha , Thanassi David , Beyaz Semir , Yang Vincent , Bliska James , Sheridan Brian S
Published: February 2023

Vγ4 T cells are an unconventional subset of T cells that protect the intestinal mucosa from pathogen invasion and capable of anamnestic responses. Yersinia pseudotuberculosis (Yptb) is a foodborne pathogen that invades through the intestinal mucosa and subverts immune function by translocation of Yersiniaouter protein (Yop) effectors into target cells. In this study, we evaluated the response of adaptive Vγ4 T cells to Yptb to determine if adaptive γδ T cells are a target of immune subversion. Tracking Yptb translocation of Yop effectors through a FRET-based reporter assay revealed targeted and preferential delivery of Yop effectors directly into adaptive γδ T cells inhibited anti-microbial IFNγ production. Using a series of recombinant Yptb deficient or defective in numerous pathogenicity genes revealed that direct subversion of the adaptive γδ T cell IFNγ response was mediated by the adhesion molecule YadA, the injectosome component YopB, and the YopJ effector. Thus, Yptb attachment and translocation of YopJ directly into adaptive γδ T cells is a major mechanism of γδ T cell subversion. RNA-Seq of adaptive γδ T cells revealed that a broad anti-pathogen gene signature was inhibited by translocation of YopJ and suggested a role of the STAT4 pathway in this process. Indeed, IL-12p40 promoted the adaptive γδ T cell IFNγ response and YopJ limited STAT4 phosphorylation levels. Importantly, circulating human Vδ2+T cells were similarly targeted and inhibited by the YopJ effector, highlighting a novel and conserved Yptb pathway that subverts adaptive γδ T cell function.
M. tuberculosis resides in permissive, lysosome-poor recruited macrophages during chronic infection
Authors: Zheng Weihao , Chang I-Chang , Limberis Jason , Ning Fei , Ernst Joel
Published: February 2023

M. tuberculosis (Mtb) infects lung phagocytes including alveolar macrophages (AM) and recruited/monocyte-derived macrophages (RM). However, little is known about the distinct roles of different cell subsets in Mtb persistence, and how they differ in controlling Mtb during chronic infection. Using fluorescent reporter strains and quantitation of live bacteria by cfu plating of sorted cell subsets, we found that AM are superior to RM in restricting and killing Mtb during chronic infection (4 wk PI). Subsequent bulk RNA-Seq of live-sorted cells from infected mice reveal that genes of the lysosome pathway are under-expressed in RM compared with AM. Using functional assays, we show here that RM have poorer lysosome function than AM. Specifically, AM not only have more lysosome content, more abundant lysosomal enzyme content and activities, but also have higher expression of proteins for lysosome acidification and more acidic lysosomes. Moreover, immunofluorescence data reveal that AM have more nuclear TFEB than RM. Nuclear translocation activates TFEB, which serves as a master regulator of lysosome biogenesis. We also found that TFEB overexpressing bone marrow-derived macrophages have enhanced abilities to kill virulent Mtb. Together, these results imply that TFEB may drive the difference of lysosome functions between AM and RM, leading to the difference in killing Mtb, and that Mtb takes advantage of intrinsically lysosome-poor RM for persistence.
Microtubules and dynein regulate human neutrophil nuclear volume and hypersegmentation during H. pylori infection
Authors: Toro Stephanie L Silva-Del , Allen Lee-Ann H
Published: February 2023

Neutrophils (polymorphonuclear leukocytes, PMNs) are heterogeneous and can exhibit considerable phenotypic and functional plasticity. In keeping with this, we discovered previously that Helicobacter pylori infection induces N1-like subtype differentiation of human PMNs that is notable for profound nuclear hypersegmentation. Herein, we utilized biochemical approaches and confocal and super-resolution microscopy to gain insight into the underlying molecular mechanisms. Sensitivity to inhibition by nocodazole and taxol indicated that microtubule dynamics were required to induce and sustain hypersegmentation, and super-resolution STED imaging demonstrated that microtubules were significantly more abundant and longer in hypersegmented cells. Dynein activity was also required and enrichment of this motor protein at the nuclear periphery was enhanced following H. pylori infection. In contrast, centrosome splitting did not occur, and lamin B receptor abundance and ER morphology were unchanged. Finally, analysis of STED image stacks using Imaris software revealed that nuclear volume increased markedly prior to the onset of hypersegmentation and that nuclear size was differentially modulated by nocodazole and taxol in the presence and absence of infection. Taken together, our data define a new mechanism of hypersegmentation that is mediated by microtubules and dynein and as such advance understanding of processes that regulate nuclear morphology.
A systemic inflammatory response primes blood monocytes in cutaneous leishmaniasis
Authors: Amorim Camila Farias , Goshu Brukawit A , Lombana Cláudia , Novais Fernanda O , Nguyen Ba T. , Carvalho Lucas P. , Carvalho Edgar M. , Beiting Daniel P. , Scott Phillip
Published: February 2023

Cutaneous leishmaniasis is a localized infection controlled by CD4+ T cells that produce IFN-y within lesions. Phagocytic cells recruited to lesions are exposed to IFN-y which triggers their ability to kill the intracellular parasites. To determine what systemic responses are occurring that might influence the disease, we performed RNA sequencing (RNA-seq) on the blood of 50 L. braziliensis-infected patients, as well as 14 healthy controls. Genes reflecting Interferon downstream activation such as GBP1-6, STAT1, IDO1, AIM2, BATF2, SOCS1 and FCGR-related genes, were upregulated in patients and functional enrichment analysis identified a transcriptional type II Interferon-stimulated gene (ISG) signature as the dominant response. An increase in monocytes and macrophages in the blood, estimated from our RNA-seq dataset, was positively correlated with this type II ISG signature. Consistent with this result, patients had circulating IFN-y in their serum. Monocytes in the bone marrow and circulating in the blood of mice infected with leishmania exhibited increased expression of Class II, further supporting the premise that there is a systemic response to infection. Together, these results suggest that there is a mechanism to limit systemic spread of the parasites, as well as enhance parasite control by pre-activating cells prior to lesion entry.
Early Th2 response to Plasmodium yoelii infection is associated with intestinal mastocytosis, bacteremia and elevated intestinal permeability.
Authors: Donnelly Erinn L. , Céspedes Nora , Garrison Sarah , Haapanen Lori , Van de Water Judy , Luckhart Shirley
Published: February 2023

The presence of bacteria in the blood or bacteremia is complication of malaria that occurs across a broad range of ages and clinical manifestations and is associated with increased morbidity and mortality, but the factors that predispose patients to developing this “leaky gut” phenotype are poorly understood. Enteric bacteria are thought to translocate from the intestinal lumen into circulation via damaged tight and adherens junctions. Mast cell (MC) activation in both humans and mice is known to disrupt epithelial junction integrity and increase intestinal permeability. Increases in intestinal MCs have been noted in both mouse and primate models of malaria. Activated MCs are potent sources of Th2 cytokines, histamine, and other factors that can influence both the innate and adaptive immune response. To better understand the temporal patterns of both the circulating and intestinal immune response to non-lethal infection, we infected C57BL/6J mice with Plasmodium yoelii yoelii 17XNL and analyzed circulating white blood cell counts, bacteria levels, cytokines, intestinal permeability to dextran and ileal MC numbers 1, 2, 4, 6, 8 and 10 days post infection. We observed an early elevation (day 4) in Th2 cytokines interleukin (IL)-4, IL-6 and IL-10. Interestingly, we also observed an early elevation in basophils and eosinophils, granulocytes associated with allergic inflammation. These findings coincided with the first appearance of MCs in the ileum and preceded the development of bacteremia (day 6) and elevations in intestinal permeability to dextran (day 8), suggesting the early Th2 response to infection is a potential driver of mastocytosis and increased permeability.
Expression of CCR2 and VEGFR1 is required for monocyte migration into Mycobacterium tuberculosis-induced lung granulomas.
Authors: Herbath Melinda , Harding Jeffrey S , Fabry Zsuzsanna , Sandor Matyas
Published: February 2023

Granulomas provide protection against Mycobacterium tuberculosis (Mtb) infection and dissemination but also contribute to long term pathogen survival in the host. Macrophages are the dominant cell type in mycobacterial granulomas, playing a central role in bacterial control. Most macrophages recruited to the lung during infection are derived from monocytes in the blood. Our lab has previously shown that continuous cell replacement is important in mycobacterial lesions and CCL2 and granuloma macrophage-produced VEGF-A are required for granuloma maintenance as blocking the action of these chemokines results in the reduction of granuloma size and number in BCG and Mtb infection.

Here we show that expression of CCR2 and VEGFR1 by monocytes is required for the migration of these cells into Mtb-induced lung granulomas. Blockade of CCR2 and VEGFR1 with Propagermanium or SU5416, respectively, reduced monocyte migration toward granuloma cells in vitro or in vivo. A 1:1:1 mixture of color-coded WT, Flt1 fl/fl x LyzMCre and CCR2−/− BM-derived monocytes was adoptively transferred into C57BL/6J recipient mice. The ratio of transferred cells in the lungs, spleen, blood, and BM was measured 1 day and 10 days after transfer. By day 10, most of the transferred cells were present in the lung parenchyma. Loss of VEGFR1 on the transferred monocytes decreased monocyte migration to the lung parenchyma dramatically while homing of CCR2−/− cells was inhibited to every tested site.

In the granulomatous lung the main source of VEGF-A is the macrophage. We are developing macrophage-targeted blockade of VEGF-A to reduce granulomatous pathology with the reduction of off-target effects.
CXCR3 gene deletion in murine cryptococcal meningoencephalitis leads to a shift in CD4-T Cell transcriptional profile away from hyperactive/cytotoxic phenotype.
Authors: Martens Jacob William Shelton , Xu Jintao , Goughenour Kristie D , Subbiah Harini , Kwak Doyun L. , Parkin Brian L. , Olszewski Michal A
Published: February 2023

Cryptococcal meningoencephalitis (CM) is the major cause of infectious neurological death worldwide, mainly affecting immunocompromised patients. CNS damage is often linked to aberrant inflammatory responses following infection. Deletion of CXCR3 in murine CM leads to improved mouse survival and diminished neuroinflammation and damage while maintaining fungal clearance. To establish how CXCR3-deletion suppressed pathological CD4 T cell responses without limiting protective responses, we analyzed transcriptional profiles of brain leukocytes isolated from WT and CXCR3KO with CM using single-cell RNA seq. Distinct clusters of CNS CD4 T-cells were found in CM mice: naïve, effector memory, activated, and exhausted T cells, as well as Th1 and Th2 clusters. The activated T cell clusters (CD69, ICOS, and 4-1BB high) exhibited a Th1-skewed phenotype (T-bet and IFN-γ high). CXCR3KO mice exhibited a marked shift away from these effector T cell clusters, with observed decreases in both memory and activated CD4 T cell phenotypes. In contrast, the naive T cell clusters were maintained in the CXCR3KO mice. The activated CD4 T cell clusters showed significant increase in expression of cytotoxic NKG7 in the WT mice compared to CXCR3KO mice. Interestingly, an IL-17A-expressing γδ-T cell cluster largely absent in the WT was increased in CXCR3KO mice.

In conclusion, CXCR3 deletion resulted in a pronounced shift away from highly activated Th1 profile toward a more moderate activated phenotype with a decrease in cytotoxicity, supporting the role of the CXCR3 axis in inducing pathological T cell phenotypes in the brain. The emergence of IL-17A-producing γδ-T cells is unexpected and deserves future studies in the context of protection versus pathology.
Identification of unique memory T cell populations associated with protection against Chlamydia trachomatis reinfection in the peripheral blood of highly exposed women
Authors: Poston Taylor , Kollipara Avinash , O’Connell Catherine , Zheng Xiaojing , Darville Toni
Published: February 2023

Chlamydia trachomatis (Ct) is an obligate intracellular bacterium that annually infects 100 million people globally and leaves 1 million women infertile, making vaccine development a priority. While repeated infections are common, they can elicit partial protective immunity. To better determine protective T cell responses elicited by natural infection to guide rational vaccine design, we performed single-cell RNA sequencing (scRNA-seq) on sorted CD3+ T cells from PBMC samples of two uninfected and four infected women, two who remained uninfected (F/U−; n=2) and two who became reinfected (F/U+; n=2) when tested at intervals over a year. We detected enrichment of two CD4 and two CD8 subpopulations in the F/U− subgroup, compared to F/U+ or uninfected controls. The CD4 T cell populations were characterized by differential expression of genes involved in cell activation, tissue homing, memory, and Th1/17 differentiation (LMNA, S100A11/10/4, ANXA1/2, LGALS1/3, TIMP1, CD29, CD161, IL7R), and the CD8 T cell populations differentially expressed markers of cell growth, activation, cytokine signaling, and cytotoxicity (CCL4/5, GRZMA/K, NKG7, CST7, HLA-DRA/B1, CD74). Both CD8 subsets expressed fewer markers of terminal differentiation, suggestive of memory and/or transitional effector phenotypes. These pilot data demonstrate that, even though Ct is mucosally restricted, differential T cell responses are detectable in peripheral blood by scRNA-seq and can improve understanding of T cell heterogeneity, activation, differentiation, and functional responses that promote protection.
Primary biodiversity data on zooplankton, macroinvertebrates, and fish from freshwater ecosystems of Uganda
Authors: Musinguzi Laban , Olokotum Mark , Nakiyende Herbert , Egessa Robert , Kiggundu Vicent , Pabire Ghandi Willy , Bassa Samuel , Nsega Monic , Kamya AshIraf , Rwezawula Philip , Lugya Jessy , Magezi Godfrey , Naluwayiro Janet , Natugonza Vianny
Published: February 2023

Effective conservation requires reliable data and information on the status of biodiversity. The conservation of freshwater biodiversity lags behind terrestrial and marine biodiversity because data and information limitations are greatest in freshwater ecosystems.  Given that freshwater ecosystems are inhabited by disproportionately more species than other ecosystems, paucity of data and information threatens many species and dependent ecosystem services. Data and information on freshwater biodiversity is limited mainly because few freshwater ecosystems are considered for regular monitoring. However, even existing data is scattered and in non-user-friendly formats, limiting accessibility and use. It is desirable to make freshwater biodiversity data and information accessible everywhere so that it attains its full potential in guiding conservation. To increase accessibility to freshwater biodiversity data in Uganda, we present 34 datasets covering three major freshwater taxa: zooplankton, macroinvertebrates, and fish within freshwater ecosystems in the country. The datasets provide occurrence records and corresponding abundance data where applicable for the three major groups. The datasets which are available through the Global Biodiversity Information Facility (GBIF) cover   a long period from 1971-2021 and have a total of 56,104 occurrence records. Of these records, 8,674 records were published in 2022. The data were mobilized from primary biodiversity surveys conducted by scientists at the National Fisheries Resources Research Institute (NaFIRRI) in Uganda. The surveys covered most of the water bodies in the country. The datasets are envisaged to increase accessibility to data for freshwater conservation research, decision making and capacity building. The data has already found use in development of conservation tools and conservation status assessments.
Epiganthus presenting as respiratory distress in a male neonate
Authors: Kumar Naveen , Ahluwalia Charanjeet , Singla Gaurav , Singla Swati
Published: February 2023

An epignathus is an extremely rare form of oropharyngeal teratoma that arises from the oral cavity, most commonly from the palate and is associated with a high mortality secondary to airway obstruction in the neonatal period. It predominantly occurs in females. Here, we are presenting a case of giant epignathus in a male baby with associated cleft palate.
Retrobulbar Hematoma in a Patient with Thyroid Eye Disease
Authors: Weng Christina Y.
Published: February 2023

A 37-year-old female with a history of thyroid eye disease presented after trauma to the right side of her face. Visual acuity was light perception and a relative afferent pupillary defect was detected. There was gross proptosis of the unruptured right globe with diffuse conjunctival hemorrhage. Computed tomography revealed an intraconal retrobulbar hematoma (RBH) with anterior herniation of the globe. The extraocular muscles were thickened, consistent with her thyroid eye disease, and her superior rectus was avulsed. The optic nerve was on stretch, but there was no evidence of transection. The patient ultimately underwent enucleation with the plan for an eventual ocular prosthesis. In RBH, blood accumulates in the retrobulbar space and can lead to an increase in intraocular pressure, optic nerve stretch, or mechanical blockage of ocular perfusion. A brief review of RBH is included to emphasize the most important aspects of diagnosis and management of this vision-threatening emergency.
Methylprednisolone pulse therapy for critically ill patients with COVID-19: a cohort study
Authors: Choi Keum-Ju , Jung Soo Kyun , Kim Kyung Chan , Kim Eun Jin
Published: February 2023

Background: The guidelines recommend the use of dexamethasone 6 mg or an equivalent dose in patients with coronavirus disease 2019 (COVID-19) who require supplemental oxygen. Given that the severity of COVID-19 varies, we investigated the effect of a pulse dose of corticosteroids on the clinical course of critically ill patients with COVID-19.Methods: This single-center, retrospective cohort study was conducted between September and December 2021, which was when the Delta variant of the COVID-19 virus was predominant. We evaluated the mortality and oxygenation of severe to critical COVID-19 cases between groups that received dexamethasone 6 mg for 10 days (control group) and methylprednisolone 250 mg/day for 3 days (pulse group).Results: Among 44 patients, 14 and 30 patients were treated with control steroids and pulse steroids, respectively. There was no difference in disease severity, time from COVID-19 diagnosis to steroid administration, or use of remdesivir or antibacterial agents between the two groups. The pulse steroid group showed a significant improvement in oxygenation before and after steroid treatment (P<0.001) compared with the control steroid group (P=0.196). There was no difference in in-hospital mortality (P=0.186); however, the pulse steroid group had a lower mortality rate (23.3%) than the control steroid group (42.9%). There was a significant difference in the length of hospital stay between both two groups (P=0.039). Conclusions: Pulse steroids showed no mortality benefit but were associated with oxygenation improvement and shorter hospital stay than control steroids. Hyperglycemia should be carefully monitored with pulse steroids.