bluuebunny/crossref_metadata_2025 huggingface dataset:
Scientific language communication within postgraduate Immunology education
Authors: Sika-Paotonu Dianne , Tait Carolyn
Published: January 2023

Language is the dominant medium through which communication occurs in tertiary education contexts. It is paramount that teachers consider the language of the classroom where learning and teaching activities occur as this is highly relevant to all students and in particular, English as Second Language (ESL) learners. The purpose of this work was to explore scientific language communication in the postgraduate learning and teaching environment for nursing, midwifery and health students undertaking Immunology classes and consider the implications for ESL learners in particular. Participant responses based on formal teaching performance and student satisfaction scores were provided by nursing, midwifery and health students in response to Immunology teaching sessions presented at postgraduate level. Formal teaching feedback from students within these postgraduate classes that included ESL students was analysed and indicated that teaching category measures for “clarity of communication” and “attitude towards students” were highly favorable. Additional feedback also indicated positive responses to in-class activities including peer-to-peer teaching and learning interactions. Student feedback includes responses including that from ESL learners and would suggest that the scientific language communication and the use of group and peer interaction activities being undertaken in this postgraduate education setting are providing support and promoting scientific language learning for all students.
Impact of antigen affinity on T cell dysfunction in solid tumors
Authors: Shakiba Mojdeh , Philip Mary , Horste Ellen , Camara Steven , Schietinger Andrea
Published: January 2023

Although tumor-specific T cells (TST) are found in human solid tumors, cancers progress, indicating that these T cells are dysfunctional. TSTs express high levels of inhibitory receptors and fail to execute effector functions; however, the regulatory mechanisms underlying TST dysfunction remain poorly defined. T cell mediated immune response is triggered by T cell receptor (TCR) binding to peptide-major histocompatibility (pMHC) complex on the surface of cells. The affinity of TCR:pMHC interaction is a critical determinant of T cell expansion and effector function in acute infections, where T cells with high affinity interactions generally show superior function. However, little is known about how TCR:pMHC affinity impacts T cells activation, induction of dysfunction and susceptibility to immunotherapy in progressing tumors. To elucidate this, we generated tumor cell lines expressing altered peptide ligands derived from SV40 large T antigen epitope I (Tag) and recognized by Tag-specific transgenic CD8 T cells (TCRTag) with varying functional avidity. We found that tumor-infiltrating TCRTag encountering low and high affinity tumor antigens were equally activated and expressed similar levels of inhibitory receptors, suggesting that even very weak TCR ligations can induce a typical “exhaustion” phenotype. Strikingly, while high affinity TCR:pMHC interactions led to complete loss of cytokine production, T cells with low affinity interactions remained functional, suggesting that TSTs with high affinity TCR:pMHC interactions enter a profound state of dysfunction. Future experiments will test the impact of tumor antigen affinity on the efficacy of therapeutic reprogramming, with important implications for immunotherapy.
Combinations of resveratrol, curcumin and cisplatin lower the apoptosis threshold in Jurkat cell lines.
Authors: Valenzuela Hector F , Grabauskas Titas , Chen Michelle , Ruiz Thomas , Kwak Yerin
Published: January 2023

Resveratrol and curcumin are natural compounds consumed for both nutritional and medicinal reasons. Resveratrol and curcumin (and corresponding analog compounds) possess potent anti-inflammatory and anti-oxidative properties. In addition, both compounds also have potent anti-proliferative effects against a number of cancer cell lines, including lymphomas. Cisplatin is a chemotherapy medication used to treat a number of cancers including lymphomas. However, cisplatin, unlike resveratrol and curcumin, have a number of harmful side effects such as neurotoxicity, nephrotoxicity, nausea and vomiting that can limit its use. The purpose of our research was to compare different combinations of resveratrol, curcumin and cisplatin as a strategy to lower the dosages of cisplatin while still inducing apoptosis on human Jurkat cells. In this study we show that inhibition of Jurkat cells is due to both inhibition of proliferation and induction of apoptosis. We used RT-PCR to also show that the down-regulation of COX-2 is also involved. These experiments support the notion that combinatory therapy is an effective strategy to treat lymphomas.
C/EBPd enables the efficient extravasation of human CCR2+ MAIT cells
Authors: Zhang Howard H , Lee Chang Hoon , Too Lily , Singh Satya , Tsang Hsinyi , Kabat Juraj , Singh Tej Pratap , Farber Joshua M
Published: January 2023

Early responses to pathogen challenge depend in part on recruitment of effector-capable T cells from blood. Human mucosal-associated invariant T (MAIT) cells are abundant, mostly CD8+, MR1-restricted, anti-bacterial T cells that could contribute to this response. However, little is known about these cells’ ability to traffic in inflammation. We found that, as compared with non-MAIT CD8+ T cells, human MAIT cells excelled at entering inflamed skin in mice. In flow chamber assays, MAIT cells were efficient at trafficking across activated human umbilical vein endothelial cells (HUVEC) due to high levels of selectin ligands, and chemokine receptors CCR6, and CCR2, which we showed were important for rolling, arrest, and transendothelial migration, respectively. We found that MAIT cells express high levels of the mRNAs for FUT7 and ST3GAL4, which are critical for synthesizing selectin ligands. We also found that expression of FUT7, ST3GAL4, and CCR6 depends on C/EBPd, a bZIP transcription factor that we discovered is expressed preferentially in MAIT cells and that binds to the FUT7, ST3GAL4, and CCR6 promoters. For MAIT cells, knockdown of C/EBPd decreased rolling and arrest on HUVEC and extravasation into inflamed mouse skin. Thus, CCR2+ MAIT cells display trafficking abilities that depend on chemokine receptors with non-redundant roles and the cooperative activities of C/EBPd-regulated genes - and exemplify effector-ready T cells that express a coordinated program enabling rapid entry into sites of inflammation.
The highly pathogenic Ebola and Nipah viruses hijack the host antiviral E3-ubiquitin ligase TRIM6 to enhance their own replication
Authors: Rajsbaum Ricardo , Bharaj Preeti , Atkins Colm , Luthra Priya , Wang Yao E , Dawes Brian E , Giraldo Maria Isabel , Park Arnold , Yen Benjamin C , Lee Benhur , Basler Christopher F , Freiberg Alexander N
Published: January 2023

The antiviral function of type-I interferons (IFN-I) requires activation of multiple signaling components, including the IκB kinase epsilon (IKKɛ). We recently reported that the E3-ubiquitin ligase TRIM6 catalyzes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, and activate IKKɛ for induction of IFN-I mediated antiviral responses. The importance of TRIM6 as an antiviral factor is highlighted by our findings that the highly pathogenic Nipah virus (NiV; Paramyxoviridae family) can inhibit IFN-I responses by targeting TRIM6. To overcome this antiviral pathway, the matrix protein of NiV promotes TRIM6 degradation, resulting in reduced levels of unanchored K48-linked polyubiquitin chains associated with IKKɛ, which in turn leads to impaired IKKɛ activation. In contrast, Ebola virus (EBOV; Filoviridae family), which antagonizes IFN-I responses via its VP35 protein, also hijacks TRIM6 to enhance its own replication, but does it primarily by directly promoting virus replication. Our data show that EBOV-VP35 is ubiquitinated by TRIM6 and promotes VP35-dependent polymerase activity. In line with these results, TRIM6-knockout cell lines and cells derived from our newly generated TRIM6−/− mouse exhibit reduced EBOV replication, even in the absence of IFN-I production. This is the first evidence indicating that TRIM6 represents a major antiviral host factor that is targeted by different viruses to promote their own replication.
Macrophage Krüppel-like factor 4 regulates response to Mycobacterium tuberculosis infection
Authors: Mazahery Claire , Chirieleison Steven , Shukla Supriya , Onwuzulike Sophia , Jain Mukesh , Boom W Henry , Abbott Derek W , Harding Clifford V
Published: January 2023

Krüppel-like factor 4 (KLF4) is a transcription factor that polarizes macrophages towards an anti-inflammatory (M2) phenotype, which suggests that it may regulate immune responses to Mycobacterium tuberculosis (Mtb), as immune evasion by Mtb may be due to an overabundance of anti-inflammatory mediators in response to infection. We hypothesize that myeloid KLF4 is permissive of immune evasion by Mtb, causing decreased control of Mtb infection. There are no prior in vivo data on the role of KLF4 in infection. Using mice with myeloid-specific knockout of KLF4 (LysMCre/CreKLF4fl/fl, abbreviated Mye-KO) we observed decreased Mtb CFU in macrophage cultures in vitro and in lungs of Mye-KO mice early in infection (day 14) relative to wild-type controls. However, these experiments also revealed complexities to the KLF4-Mtb relationship. Despite their improved control of early infection, at later time points Mye-KO mice developed worsened clinical disease features, such as wasting, which suggests that the loss of KLF4 results in pathologic immune state. In addition, we found discrepancy between KLF4 RNA and protein expression during Mtb infection, suggesting that KLF4 is regulated at a posttranslational level during infection. We are designing an in vitro system in which to study the molecular mechanisms of KLF4 regulation and activity in macrophages. Our data indicate that KLF4 plays an important role is regulating immune responses to Mtb with the potential to both diminish host defense mechanisms and repress host-damaging immune mechanisms.
Vaccinia virus host-range factor C7 has dual inhibitory functions in type I IFN production and signaling
Authors: YANG NING , Shuman Stewart , Deng Liang
Published: January 2023

Vaccinia virus C7 protein is an important host-range factor for vaccinia virus life cycle in mammalian cells. C7L homologs are present in almost all of the poxviruses that infect mammalian hosts. Type I IFN plays an important role in host defense of viral infection, and yet, the role of C7 in immune modulation of the IFN pathway is unclear. We have previously reported that the highly attenuated modified vaccinia virus Ankara (MVA) infection of conventional dendritic cells (cDCs) induces type I IFN via the cGAS/STING/TBK1/IRF3 pathway. In this study, we find that ectopic C7 expression blocks STING, TBK1, or IRF3-induced IFNB and ISRE (interferon stimulated response element) promoter activation. Murine or human macrophage cell lines that overexpress C7 have blunted innate immune responses to DNA or RNA stimuli, or the infection of DNA or RNA viruses. Overexpression of C7 also attenuates ISG gene expression induced by IFN-b treatment. MVA with deletion of C7L (MVAΔC7L) infection of cDCs induces higher levels of type I IFN than MVA. C7 blocks IFN-b-induced Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway via preventing Stat2 phosphorylation. C7 directly interacts with stat2 as demonstrated by co-immunoprecipitation studies. Taken together, our results provide evidence that C7 has dual inhibitory roles in type I IFN production and signaling.
mMGL receptor plays an important role to macrophage activation during Trypanosoma cruzi infection
Authors: Rodriguez-Sosa Miriam , Juarez-Avelar Imelda , Vazquez-Mendoza Alicia , Rodriguez-Hernandez Mario Alberto Tonathiu
Published: January 2023

The C-type lectin receptor (MGL) is exclusively expressed on macrophages (MΦs) and dendritic cells (DCs), recognizes glycosylated antigens, particularly galactose (Gal) and N-acetylgalactosamine (GalNAc), that are present in tumors, fungi and viruses. However, little is known the role of MGL in the interaction with parasites. We evaluated the role of murine MGL (mMGL) in the activation and functionality of MΦs in Trypanosoma cruzi (T. cruzi) infection, a parasite that has molecules glycosylated rich in Gal and GalNAc in its membrane.

Peritoneal MΦs from C57BL/6 WT and mMGL nockout (mMGL−/−) mice were infected in vitro with epimastigotes of T. cruz,i Qro strain (10000 parasites). The percentage of infected MΦs and the number of internalized parasites/MΦs was analyzed by microcopy. Cytokines TNF-α, IL-12 and IL-10 were determine in supernatants by ELISA. The trypanocidal capacity of MΦs was determined by the incorporation of tritiated thymidine ([3H] TdR). Expression of TLR-4, TLR-2 and mannose receptor (MR) in MΦs stimulated in vitro with T. cruzi antigen was analyzed by flow cytometry.

mMGL−/− MΦs were higher infected with more parasites per MΦ and had a reduced capacity to eliminate the T. cruzi, these were associated to lower levels of TNF-α, TLR-4 and MR expression compared to WT MΦs. These results demonstrate that absence of mMGL favors susceptibility to infection with T. cruzi, suggesting a regulatory role of mMGL on the expression of other important receptors in the immune response to T. cruzi infection.
Myeloid and plasmacytoid dendritic cell subsets are differentially regulated by the pregnancy hormone human Chorionic Gonadotropin
Authors: Schumacher Anne , Sauss Karoline , Ehrentraut Stefanie , Zenclussen Ana
Published: January 2023

Dendritic cells (DCs) are critically involved in the establishment and maintenance of fetal tolerance. However, the precise mechanisms underlying their regulation during pregnancy are still not defined. The placenta-derived hormone human Chorionic Gonadotropin (hCG) possesses immune modulatory properties and is commonly used in artificial reproductive techniques (ART). Here, we analyzed the effect of placenta-derived hCG and two hCG preparations on different DC subsets.

After isolation from peripheral blood of non pregnant women, DCs were stimulated with LPS to induce maturation. In parallel, DCs were co-cultured with placenta-derived hCG (JEG-3 cells), with urine-derived hCG or recombinant hCG. The total number and maturation state of two myeloid DC (MDC1; MDC2) and one plasmacytoid DC subset (PDC) was evaluated. Additionally, the levels of IL-6, IL-8, IL-10, IL-12 and TNF were determined in co-culture supernatants. In all experiments, unstimulated DCs and stimulated DCs cultured alone served as controls.

JEG-3 impaired the LPS-induced increase of total MDC1 while both hCG preparations hindered the increment of total MDC1 and MDC2. Moreover, JEG-3 interfered with PDC and MDC1 maturation while both hCG preparations hampered maturation of all DC subsets. However, significant effects could only be observed on the MDC1 subset. Cytokine assays revealed that JEG-3 and both hCG preparations impaired LPS-induced secretion of all analyzed cytokines.

Our results suggest that hCG from distinct sources regulates DC subsets in a different manner. This information does not only contribute to our understanding of a differential regulation of various DC subsets during pregnancy but is also relevant for the use of hCG preparations in ART.
Simultaneous ablation of natural killer cells and mast cells leads to poor uterine vascularization that compromises fetal-well being.
Authors: Zenclussen Ana , Meyer Nicole , Schumacher Anne , Pollheimer Jürgen
Published: January 2023

Once pregnancy is established, uterine spiral arteries (SA) need to undergo dramatic remodeling to ensure a consistent blood supply to the fetus.

Here, we studied the single and joint participation of natural killer cells (NKs) and mast cells (MCs) in SA remodeling and pregnancy outcome and investigated the underlying mechanisms. We show that NK deficiency or their depletion arrested SA remodeling without affecting pregnancy. MC absence resulted in abnormally remodeled SAs and intrauterine growth restriction (IUGR). Combined absence of NKs and MCs dramatically affected SA remodeling, and impaired fetal growth. Despite abnormal Doppler measurements and dramatic IUGR, maternal maternal blood pressure was not affected. We found that a-chymase mast cell protease (Mcpt) 5 mediates apoptosis of uterine smooth muscle cells, a key feature of SA remodeling. Additionally, we report a previously unknown source for Mcpt5: uterine (u) NKs. We confirmed the importance of Mcpt5 by employing mice with selective deletion of Mcpt5+ cells: pregnant females presented un-remodeled SAs and growth-restricted progeny. The human a-chymase CMA1, phylogenetic homolog of Mcpt5, stimulated the ex vivo migration of human trophoblasts, a pre-requisite for SA remodeling.

Our results show that chymases secreted by uMCs and uNKs are pivotal to the vascular changes required to support pregnancy. Understanding the mechanisms underlying pregnancy-induced vascular changes is essential for developing therapeutic options against pregnancy complications associated with poor vascular remodeling.
Cutaneous commensal bacteria drive an unconventional T cell response that accelerates wound healing
Authors: Linehan Jonathan L , Belkaid Yasmine
Published: January 2023

Our laboratory recently demonstrated that skin-resident commensals control steady state immunity and host protective responses to cutaneous infection. However, little is known about the mechanisms by which this occurs, including which arms of the immune system are involved. S. epidermidis (S.epi), a common human skin commensal, promotes a robust Interleukin-17A+ (IL-17A+) CD8+ T cell response when applied to the skin of mice. We investigated whether these cells were restricted to MHCIa or MHCIb molecules. Surprisingly, S.epi evoked CD8+ T cells produced IL-17A in the presence of S.epi loaded KbDb−/− dendritic cells (DCs), but not H2-M3−/− DCs, indicating that these cells are restricted to the MHCIb molecule H2-M3. We previously showed that S.epi induced CD8+ T cells share many traits with skin resident memory T (TRM) cells. Strikingly, RNA-seq analysis identified a profile for IL-17A+ CD8+ T cells distinct from TRM cells, including upregulated transcripts typically associated with tissue repair. Supporting the idea that these cells may control skin physiology, we found that pre-exposure of mice with S.epi led to significant differences in the early events of wound healing, including accelerated wound closure. We believe these data are the first formal demonstration that MHCIb molecules can function to present commensal derived antigens to T cells and are direct mediators in the interaction between host and microbiota. Additionally, these results reveal a T cell response that couples antimicrobial function with tissue repair, a form of immunity not previously appreciated. These findings have important implications for our understanding of tissue immunity, wound healing, and tissue specific immune responses to the microbiota.
Differential localization of distinct CD4 T cell subsets in the female genital tract during the menstrual cycle is regulated by CCR5
Authors: Kohlmeier Alison , McMaster Sean , Li Zheng-Rong , Radzio-Basu Jessica , Haddad Lisa B , Sheth Anandi N , Cong Mian-er , Dinh Chuong , Martin Amy , Hayward Sarah L , Ofotokun Igho , Antia Rustom , Garcia-Lerma J. Gerardo , Kohlmeier Jacob E
Published: January 2023

CD4 T cells are essential for optimal immune responses against sexually transmitted infections, yet the composition of the CD4 T cell pool localized in the female genital tract (FGT), and the impact of the menstrual cycle on the CD4 T cell population dynamics in the FGT, remains poorly defined. Here we show the FGT CD4 T cell pool is comprised of distinct subsets of memory CD4 T cells based upon trafficking profile, anatomic regionalization, and effector responses. Based on expression of trafficking and tissue retention markers, FGT CD4 T cells are comprised of both migratory memory (TMM) and resident memory (TRM), with TMM expressing a broad array of trafficking markers that would enable their ability to perform immune surveillance in diverse tissues. Furthermore, these subsets differ in both their regionalization throughout the FGT and in their cytokine responses, notably IL-2 production. The frequency of TMM in the FGT increased during the luteal phase of the menstrual cycle and correlated with increased CCR5 expression on circulating memory CD4 T cells. Using a dual adoptive transfer mouse model, we observed that CCR5-mediated trafficking was required for the localization of memory CD4 T cells to the luminal surface of the FGT. In addition, to test cycle influence on T cell trafficking into the FGT, we administered a CCR5 antagonist to pigtail macaques at different stages of the menstrual cycle and found that inhibition of CCR5 signaling selectively reduced TMM numbers solely at luteal time points. Together, these data provide broader insight into role that distinct tissue resident CD4 T cell subsets play in barrier defenses at the FGT and also demonstrate a role for the menstrual cycle in modulating immune protection through CCR5 signaling.
TGFBR1M318R+/−: A monogenic presentation of eosinophilic esophagitis.
Authors: Laky Karen , Kinard Jessica L. , Guerrerio Anthony , Frischmeyer-Guerrerio Pamela
Published: January 2023

Eosinophilic esophagitis (EoE) is a chronic immune-mediated disease characterized by esophageal dysfunction and accumulation of tissue eosinophils, T cells, and mast cells. The exact causes of EoE are not known. Sequence variants associated with increased EoE susceptibility have been identified, suggesting that there can be a genetic component to the disease. Several of the genes encode proteins involved in TGFβ signaling. Patients and mice heterozygous for a mutation in the kinase domain of TGFbR1 (TGFBR1M318R+/−) exhibit cardiovascular, skeletal, and immune manifestations of Loeys-Dietz syndrome (LDS). Immune symptoms include elevated serum IgE and a propensity to develop EoE. We used TGFBR1M318R+/− mice to determine which cells are responsible for the initiation and perpetuation of EoE. Current dogma posits that EoE occurs when Th2 cells are stimulated by inhaled or ingested antigens to secrete cytokines that then recruit and stimulate eosinophils and mast cells. Thus we were surprised to find that RAG2−/−R1 mice developed disease, illustrating that EoE associated with the TGFBR1M318R mutation was not lymphocyte-dependent. Bone marrow (BM) chimeras were made to confirm that altered TGFβ signaling in radio-resistant cells was sufficient to cause EoE. Lethally irradiated R1mice reconstituted with WT BM developed EoE. In contrast, eosinophils and IgE were not elevated in WT mice reconstituted with R1BM. We looked for other potential sources of type 2 cytokines and discovered that type 2 innate lymphoid cells (ILC2) were increased in the esophagus of R1 mice. We propose that altered TGFβ signaling in epithelial cells leads to EoE by stimulating ILC2 to proliferate and secrete cytokines that result in accumulation of eosinophils.
Distribution and phenotype of HIV-specific CD8 T cells in situ in the rectal mucosa of HIV+ elite controllers
Authors: Li Shengbin , Kovacs Katalin , Kiniry Brenna , Wagstaff Reece , April Ferre , Hunt Peter , Somsouk Ma , Shacklett Barbara L. , Skinner Pamela J
Published: January 2023

Mucosal tissues including the gastrointestinal tract are important reservoirs for HIV replication. HIV-specific CD8 T cells are essential in controlling HIV infection. In order to gain insights into HIV-specific CD8 T cells in elite controllers (EC), we characterized this important cell population in rectal biopsies from EC using in situ tetramer staining combined with immunohistochemistry followed by quantitative analysis. Results showed tetramer+ HIV-specific CD8 T cells distributed unevenly throughout the rectal mucosa including within lymphoid aggregates. Frequencies of tetramer+ HIV-specific CD8 T cells ranged from 2 to 25 cells/mm2. A mean of 55% tetramer+ HIV-specific CD8 T cells expressed the effector protein perforin. Among the perforin+ HIV-specific T cells, the majority expressed low to medium levels of perforin and relatively few expressed high levels. In addition, we also observed a subset of perforin+ HIV-specific CD8 T cells in which perforin was located exclusively within the cell membrane, possibly representing a novel subset of armed resident or effector memory cells. Almost all tetramer+ HIV-specific CD8 T cells in the rectal mucosa were negative for Ki67, a marker for activation and proliferation (mean of 96%); many were PD-1+, a marker for recent T cell stimulation or exhaustion (mean of 59%), and some expressed CD103, a marker for a subset of resident memory T cells (mean of 6%). Taken together, these findings indicate that EC maintain diverse populations of memory HIV-specific CD8 T cells distributed throughout the rectal mucosa including within lymphoid aggregates and suggests that these cells are likely continually being exposed to antigen, but not dividing.
Lymphocyte activation gene-3 regulates mitochondrial biogenesis and metabolism of naive CD4+ T cells
Authors: Previte Dana Michelle , Menk Ashley V. , Wright Rebecca H. , Coudriet Gina M. , Delgoffe Greg M. , Piganelli Jon D
Published: January 2023

Lymphocyte Activation Gene-3 (LAG-3) is an inhibitory receptor expressed on the surface of various immune cells, including CD4+ T cells. While LAG-3 is more frequently associated with T cell exhaustion and regulatory T cells, it is also a negative regulator of T cell homeostatic expansion. Current interest in how cellular metabolism dictates immune cell function and fate has elucidated that CD4+ T cell function is tightly coupled to metabolism. Therefore, given that LAG-3 is a regulator of CD4+ T cell homeostatic expansion, and the ability to proliferate is highly dependent upon metabolism, we hypothesized that LAG-3 regulates the metabolic profile of naïve CD4+ T cells. Naive OT.II T cells were isolated from wildtype and LAG-3−/− animals, and analyzed by Seahorse Flux Analyzer. LAG-3−/− OT.II cells demonstrated significantly increased basal respiration, spare respiratory capacity, and aerobic glycolysis, as compared to wildtype OT.IIs, indicating an enhanced metabolic profile. Further analysis indicated that LAG-3-deficient OT.IIs displayed increased mitochondrial biogenesis via enhanced signaling through the AMPK/Sirt-1 pathway. In an adoptive transfer model of homeostatic expansion, LAG-3−/− OT.II cells expanded to a greater extent and continued to demonstrate an enhanced metabolic profile as compared to WT OT.IIs. This proved to be extrinsically regulated, as LAG-3 blockade in WT recipients recapitulated similar results to LAG-3−/− T cells. These results demonstrate that LAG-3 is critical for actively maintaining metabolic quiescence in naïve CD4+ T cells. Future directions include investigating the influence of LAG-3 and its metabolic regulation in the context of autoimmunity.
Single-cell analysis of CCR6+ human Th cells reveals varying degrees of the type-17 phenotype, ranging from cells co-expressing to those fully suppressing opposing pathways
Authors: Singh Satya , Edara Nithin , Zhang Hongwei , Chen Jinguo , Farber Joshua
Published: January 2023

Previously, we found that human type-17 Th cells from blood are fully contained within the CCR6-expressing subset. We also found positive correlations between levels of expression of CCR6 and levels of other type-17 proteins such as IL-17A/F, IL-22, and CCL20, □□□ negative correlations of CCR6 expression with that of genes/proteins associated with non-type-17 (Th1, Th2, TFH) pathways. In the present study, analysis of single Th cells expressing various levels of CCR6 using cell sorting followed by RT-PCR revealed that 1) patterns of expression of individual type-17 genes differed, being either binary (all-or-none-like) or graded, 2) expression levels of type-17 genes were positively correlated with each other and negatively correlated with genes associated with non-type-17 lineages, 3) cells could be identified that co-expressed genes for both type-17 and non-type-17 lineages, and 4) these “multi-lineage” cells expressed intermediate/low as opposed to high levels of the lineage-specific genes. Our data suggest that acquisition of the type-17 (and non-type-17) phenotype(s) is not all or none, but rather that cells occupy many intermediate states in forming a highly heterogeneous memory population, that genes defining “opposing” lineages can be co-expressed, and that expression of genes associated with opposing lineages is extinguished in cells at the far end of the type-17 spectrum. These findings are consistent with pathways of human Th differentiation in vivo that allow for the co-expression of multiple potentials, each to a limited degree, early on, but that subsequently narrow as a single phenotype becomes fully expressed.
Novel function for DEPTOR in CD4+ T cell activation and differentiation
Authors: Wedel Johannes , Bruneau Sarah , Liu Kaifeng , Laplante Mathieu , Briscoe David M
Published: January 2023

In these studies we find that DEPTOR, a modulator of mTOR signaling, is expressed in CD4+ T cells and is markedly reduced upon T cell activation. We isolated CD4+ T cells from a doxycycline-inducible transgenic mouse (rtTA+/+DEPTOR+/+ or ‘iDEP’) and, by Western blot, find that its expression is associated with an inhibition of mTORC1 activity. To test if DEPTOR functions in the modulation of both Teff and Treg cell biology, iDEP naive CD4+CD25− T cells were activated in standard Th/Treg-polarizing conditions, and Th differentiation was analyzed by qPCR and ELISpot. Induced DEPTOR favored IFN-γ production in Th1 polarized cells but dramatically inhibited IL-4 (P<0.01, n=3)/IL-10 (P<0.05, n=3) and IL-17A (P<0.05, n=3) in Th2 and Th17 cultures respectively. In contrast, in iTreg polarizing cultures, Foxp3 expression was not influenced by DEPTOR (85.5±5.3% vs. 84.3±8.1% Foxp3+ cells, n=4; P=n.s.). However, in in vitro suppression assays, induced DEPTOR augmented iTreg function (AUC: 228.8±27.5 vs. 322.0±32.9, n=3; P<0.05). Also, DEPTOR overexpressing iTregs retain higher frequencies of Foxp3 after re-stimulation with anti-CD3/CD28 (46.1±6.0% vs. 9.9±5.8% Foxp3+ cells on day 7, n=3; P<0.001). To confirm that DEPTOR functions in vivo, iDEP CD4+ T cells were adoptively transferred (day 2) into Rag2γc−/− recipients (H-2b) of fully MHC mismatched BALB/c (H-2d) heart transplants. Induced DEPTOR within transferred CD4+ cells resulted in a significant prolongation of graft survival (37 days, n=5 vs. 16 days, n=6; P<0.01).

Collectively, these findings identify CD4+ T cell DEPTOR as a novel cell intrinsic protein that functions to regulate Teff differentiation and Treg function in vitro, and to promote immunoregulation in vivo.
Natural ThPOK− CD4 cytotoxic T lymphocytes : Thymic development of a new distinct self-specific CD4 T cell population.
Authors: Thiault Nicolas , Mushtaq Mohd.Husain , Iwaya Hitoshi , Verstichel Greet , Cheroutre Hilde
Published: January 2023

The lineage commitment to either CD4 or CD8 T cells is made in the thymus, where thymocytes with specificity for MHC-II molecules differentiates into the CD4 “helper” linage whereas ones specific for MHC-I molecules commit to the CD8 “cytotoxic” lineage. The molecular regulation is controlled by the action of key transcription factors. Notably ThPOK, which promotes the CD4 fate and suppress the cytotoxic fate of CD4 T cells. Recently, we made a major breakthrough and showed that these lineages is not fixed and the mature peripheral CD4 T cells have the plasticity to lose ThPOK expression and reprogram to ThPOKneg CD4 CTL (cytotoxic T lymphocytes) marked by the expression of CD8a, Granzyme B and potent killer capacity.

Investigating the origin of CD4 CTL, we observed in ThPOK fate maping mouse a strong proportion of CD4 CTL that never expressed ThPOK, suggesting that some CD4 CTL are natural cytotoxic cells that develop in the thymus. Based on a recent works allowing the analysis of mature CD4 cells in the thymus, we observed a substantial number of mature CD4 T cells that do not express ThPOK and display an active-like phenotype. Moreover, once injected, thymic ThPOKneg mature CD4 T cells remain ThPOKneg in the periphery and start to acquire CD8a expression demonstrating that natural CD4 CTL arise from the thymus. We also demonstrated that this population requires a strong TCR signal during their development, suggesting that natural CD4 CTL have a repertoire specific for self-antigens.

We have identified a new distinct population of mature CD4 T cells that develop in the thymus: the natural cytotoxic CD4 T lymphocytes. We also established mechanism underlying these process by demonstrating that CD4 CTL needs a strong TCR signal for their differentiation.
Memory T cells contribute to early pathogen clearance in a site-specific manner in the absence of cognate antigen
Authors: Maurice Nicholas Joseph , Berkson Julia D , Prlic Martin
Published: January 2023

Memory CD8+ T lymphocytes are typically considered to contribute to host immunity when they reencounter their specific antigen; however, in the absence of cognate antigen, inflammatory signals can elicit the rapid expression of Granzyme B and Interferon-γ in memory CD8+ T cells. This is referred to as “bystander activation” and occurs during the early stages of infection, which are dominated by innate immune responses. We and others have demonstrated that bystander-activated cytotoxic T lymphocytes (BA-CTL) can kill target cells in an NKG2D-ssdependent, TCR-independent manner early after infection. This suggests that memory T cells can employ innate-like mechanisms for rapid TCR-independent responses to infection. Here we report that antigen non-specific BA-CTL display clustering and increased functionality at sites of splenic pathogen replication during WT Listeria monocytogenes (LM) infection. These clusters at early time-points closely resemble those of antigen-specific cells during primary and recall responses against recombinant LM. We describe the mechanisms driving this early activation and define distinct stages of this innate-like effector program. We propose that the purpose of recruiting BA-CTL to sites of infection and eliciting these innate-like functional capacities is to limit early pathogen replication.
Characterization of CD31 expression on murine and human neonatal T lymphocytes during development and activation
Authors: Fike Adam J , Nguyen Linda T , Kumova Ogan K , Carey Alison J
Published: January 2023

CD31, expressed by the majority of the neonatal T cell pool, is involved in modulation of T-cell receptor (TCR) signalling by increasing the threshold for T cell activation. Therefore, CD31 could impact neonatal tolerance or immune responses during infection. Cells were harvested from murine neonates at various days post-birth, human late preterm and term cord blood and adult peripheral blood. The number of CD31 expressing CD4+ and CD8a+ T cells increase in the neonatal mouse over the first week of life, which is consistent with previous work demonstrating an increase in CD31 expression across human gestation. Human samples were activated in vitro over a five-day period to simulate acute inflammation similar to chorioamnionitis or early onset sepsis. The frequency of human CD31+ CD4+ and CD8a+ T cells decrease upon activation, with infants having a lower frequency of CD31+ cells compared to adults. In order to further evaluate the regulation of CD31 during acute infection, murine neonates at 3 and 7 days of age were infected with influenza. There was an increased number of naïve and activated CD31+ T lymphocytes at the site of infection at 6 and 9 days post-infection in the 3-day old neonates (p<0.05), as compared to 7-day old. However, the opposite is true in the periphery; mice infected at 7-days of age have an increased number of activated CD31+ T lymphocytes in the spleen at 9 days post-infection (p<0.05). Trafficking differences of CD31+T lymphocytes to the site of infection suggest a differential regulation in younger mice which could contribute to the dampened adaptive immune response in neonates. Further murine and human mechanistic studies which examine CD31+ cytotoxic T lymphocyte function at the site of infection are needed.
Cholinergic stimulation enhances intestinal antimicrobial activity and prevents systemic dissemination of pathogenic bacteria
Authors: Fernandez-Cabezudo Maria J , Al-Barazei Ray , Bashir Ghada , Mohamed Yassir A. , Qureshi Mohammad M , al-Ramadi Basel B
Published: January 2023

Inflammation constitutes an important component of host defense against pathogenic bacteria. This innate immune response is crucial for clearance of pathogens and survival of the host. However, unrestrained inflammatory reaction may result in systemic manifestations with dire consequences to the host. The extent of activation of the inflammatory response is tightly regulated through immunological and neural pathways. We have previously demonstrated that cholinergic stimulation, by administration of acetylcholinesterase inhibitors, confers enhanced protection in experimental animals orally infected with virulent Salmonella typhymurium. In this study, we investigated the mechanism by which AChE inhibition modulates the immune response to Salmonella infection. Enhanced host survival following oral infection correlated with significantly reduced bacterial dissemination to target organs, including livers and spleens. This protection was not due to increased gut motility or rapid bacterial clearance from the gastrointestinal tract. Moreover, protection was lost when the animals were infected systemically, suggesting that acetylcholine-mediated protective effect was mostly confined to gut mucosal tissue. In vivo imaging demonstrated more localized infection and delay in bacterial dissemination into systemic organs in mice pre-treated with AChE inhibitors. Morphological analysis of small intestine (ileum) showed that AChE inhibition induced degranulation of goblet cells and Paneth cells, two specialized secretory cells involved in innate immunity. Our findings demonstrate a crucial pathway between neural and immune systems that acts at the mucosal interface to protect the host against invading pathogens.
SHP1 alters TCR signal strength and metabolism
Authors: Martinez Hunter , Martinez Ryan J , Kolawole Elizabeth M , Evavold Brian D
Published: January 2023

Recently, we have shown thymocytes lacking SHP1 have decreased cell numbers post selection for both CD4 SP and CD8 SP. This thymocyte selection defect led to decreases in tetramer positive precursor numbers in the periphery. Specifically we analyzed CD8 T cells for LCMV GP33–41:Kd and CD4 T cells for MOG35–55:IAb. Immunization with LCMV Armstrong revealed about 50% reduction of antigen specific T cells, both CD4+ and CD8+. Even though SHP1 deficient T cells have reduced numbers, they exhibit superior proliferation as observed by cell trace violet coupled with increased death. Though SHP1 has many implications in T cell signaling, a novel aspect is impacting metabolism as the SHP1 deficient T cells display increased metabolic activity. Naïve SHP1 deficient CD8 T cells exhibit increased mitochondrial respiration. During expansion, these T cells maintain increased mitochondrial respiration compared to wildtype controls. Comparatively, lack of SHP1 also causes an increase in glucose uptake throughout expansion. Altogether, SHP1 appears to impact TCR mediated signal threshold which leads to increased T cell metabolism in peripheral tissues.
Assessing the immunomodulatory potential of therapeutic agents in cynomolgus macaque cytotoxic T lymphocytes.
Authors: Frank Brendon , Guerrero Abraham , Lebrec Herve , Balazs Mercedesz
Published: January 2023

The preclinical evaluation of therapeutic agents contributes to the understanding of associated risks and benefits. The non-human primate, and notably the cynomolgus macaque, is often a species of choice for such pharmacological and toxicological assessments. The degree of immunomodulation associated with these agents, intended or not, is a critical point to consider in that context. While there are tools to immunophenotype and enumerate effector T cells, the knowledge gap surrounding the functional characterization of cytotoxic T lymphocytes (CTL) persists. Here we present a multi-parametric cynomolgus macaque CTL functional assay. By using an EGFR expressing target cell line co-cultured with cynomolgus peripheral blood mononuclear cells (PBMC) and an EGFR Bispecific T Cell Engager (BiTE®), we were able to redirect lysis in a target-specific manner. In this assay we simultaneously monitored cytolytic potential (CD107a) and cytokine secretion (IFN-γ and TNF-α) of effector CTL against target cell growth. Overall our results demonstrate the ability to assess cynomolgus CTL function under known immunosuppressive agents, thereby enabling a multi-parametric approach of monitoring the impact of investigational drugs on an important component of the immune system.
Contribution of NKT follicular helper cells to T-dependent and T-independent humoral immunity against bacterial toxins and polysaccharides
Authors: Lang Mark L , Lang Gillian A. , Rampuria Pragya , Shrestha Binu , Ballard Jimmy D
Published: January 2023

Activation of CD1d-restricted Natural Killer T (NKT) cells with the CD1d-binding glycolipid α-galactosylceramide (α-GC), promotes humoral immunity against co-administered T-dependent and T-independent antigens. The discovery of a Bcl6hi/CXCR5+/PD-1hi, IL-21-secreting ‘follicular helper’ NKT subset (NKTfh) led to the hypothesis that NKTfh cells promote humoral immune responses to vaccine candidates including bacterial toxins and surface polysaccharides. We used Cre-Lox technology to ablate Bcl6 expression by CD4-expressing cells in mice and examine the impact on NKT-driven humoral immunity to Clostridium difficile-derived Toxin B (TcdB) and surface carbohydrates (PSI, II, and III) as well as model polysaccharides (Ficoll). In the CD4Cre;Bcl6fl/floffspring, we observed ablation of Bcl6hi/CXCR5+/PD-1hi NKT cells without depleting the parent NKT population. Adoptive transfer of NKT cells from B6 donors capable of NKTfh differentiation demonstrated a contribution to the T-dependent TcdB-specific antibody response. As expected, NP-conjugated Ficoll produced a strong NP-specific IgM and IgG3 response when administered alone, and selective class switch to IgG1 was observed when α-GC was co-administered. In NKTfh cell-ablated mice, the IgG1 response was abolished while the IgM and IgG3 responses remained intact. Experiments are underway to determine if this result will be replicated using C. difficile-derived polysaccharides. In conclusion, NKTfh differentiation driven by glycolipid adjuvants can enhance and/or broaden humoral immune responses against pathogen-derived T-dependent and T-independent antigens.
ADAM10 regulates the ICOS:ICOSL axis
Authors: Lownik Joseph Cornelius , Luker Andrea , Martin Rebecca , Damle Sheela , Conrad Daniel H
Published: January 2023

The interaction between inducible costimulator (ICOS) and its ligand (ICOSL) is essential for proper T-dependent humoral immune responses, especially follicular helper T cell (TFH) development. We have demonstrated that A Disintegrin and Metalloproteinase 10 (ADAM10) is the primary and physiological sheddase of ICOSL. Using an in vivo system in which ADAM10 is deleted only on B cells (ADAM10B−/−) leads to high ICOSL levels which in turn causes downregulation of surface T cell ICOS expression through internalization of ICOS. In an unimmunized state, ICOS is internalized in ADAM10B−/−mice and is preferentially shuttled to the lysosomal compartment for degradation. We have recapitulated these findings in an in vitro system, showing that upon ligation, ICOS is rapidly internalized and is shuttled to endosomal recycling or lysosomal degradation, depending on availability of TCR costimulation. Thus, elevated ICOSL in the absence of TCR stimulation results in loss of ICOS and recapitulates the phenotype of an ICOS knockout with severe defects in T follicular helper (TFH) development and Th2 polarization, as well as enhanced Th1 and Th17 immune responses. Blockade of ICOSL in ADAM10B−/−mice rescues T cell ICOS surface expression and restores both TFH numbers and antibody production. Overall, we propose a novel regulation of the ICOS:ICOSL axis, with ADAM10 directly regulating ICOSL which in turn regulates ICOS levels.
Unique properties of GC B cells permit optimal BCR affinity discrimination
Authors: Kwak Kihyuck , Quizon Nicholas , Sohn Haewon , Manzella-Lapeira Javier , Spillane Katelyn , Holla Prasida , Lu Jinghua , Tolar Pavel , Pierce Susan K.
Published: January 2023

Upon encountering pathogens, B cells within lymphoid organs enter specialized micro-environments of germinal centers (GCs). In the dark zones (DZ) of GCs, B cells proliferate and undergo somatic hypermutation, and subsequently migrate to the light zone (LZ) of the GC where selection of B cells that express the highest affinity B cell receptors (BCRs) occurs. Affinity selection is driven by the ability of BCRs to signal and extract antigen from the surfaces of follicular dendritic cells. Here we provide evidence that human tonsil LZ GC B cells are inherently better able to undergo affinity selection as compared to naïve tonsil B cells. LZ GC B cells engaged membranes that contained F(ab’)2 anti-light chain as a surrogate antigen through highly dynamic F-actin-containing pod-like structures that concentrated BCRs at their contact points. Using DNA-based mechanical force nano-sensors we observed robust pulling forces localized to the contact sites of the pods with the membrane. In contrast naïve B cells formed flat stable contacts with the membranes and showed only defuse weak pulling forces. Using antikappa antibodies of low (KD=3.9×10−7) versus high (KD=2.4×10−9) affinities as surrogate antigens, we show that LZ GC B cells were able to discriminate between these and responded only to the high affinity antigen. In contrast, naïve B cells responded equally to both antigens. Both LZ GC B cells and naive B cells were able to extract and internalize membrane-bound antigen, however, only in LZ GC B cells was the subsequent trafficking of the antigen to acidic intracellular compartments for processing highly dependent on the amount of antigen internalized. Thus, LZ GC B cells appear to be intrinsically programmed for optimal affinity selection.