bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene expression profiling of samples of limited RNA amounts by the NGS application whole transcriptome AmpliSeq-RNA on the Ion Torrent platform offers straightforward, fast, and affordable analysis of human T cell subpopulations.
Authors: Raulf Friedrich , Allard Cyril , Delucis-Bronn Corinne , Angevaare Eline , Vedrine Corinne , Boesch Danielle , Brinkmann Volker
Published: January 2023

The diversity of T cell subpopulations demands a growing number of biomarkers to be studied from small samples. To bridge the gap between multicolor flow cytometry or classical qPCR and RNA-Seq, we applied highly multiplexed whole transcriptome AmpliSeq-RNA with 20,803 genes to perform sensitive and specific gene expression profiling (GEP). This study analyzed untouched primary human naïve and memory T cells and compared results to microarrays.

Method

8 T cell subsets were isolated by FACS from 4 healthy donors without restimulation: CD4+ or CD8+ naïve (CCR7+CD45RA+), central memory (CCR7+CD45RA−), effector memory TEM (CCR7−CD45RA−) and TEMRA (CCR7−CD45RA+). AmpliSeq transcriptome human gene expression kit (Thermo Fisher) generated barcoded libraries from total RNA. 8–10 samples multiplexed per Ion 540 chip were sequenced on an Ion S5 System. Samples were also analyzed by Affymetrix HG-U133 Plus 2 genechips.

Results

The resulting Excel matrix of normalized reads [RPM = reads per million reads] had a dynamic range of more than 5 log units. Biological replicates of T cell subsets showed high correlation with r ≥ 0.925. Technical replicates (same libraries) demonstrated high reproducibility with r ≥ 0.995. A high concordance with genechip data were found for expression pattern of analyzed markers. For several very high or low abundant genes the AmpliSeq-RNA data were more sensitive and more specific than the hybridization-based genechip data.

Conclusion

AmpliSeq-RNA (counting PCR products by NGS) is an interesting alternative to perform straightforward GEP. The analysis of phenotypically well-defined T cell subsets without artificial in vitro stimulation allows more precise determination of in vivo relevant functions.
iPair and iPair+: cognate pairing of human TCR alpha and beta V-regions and phenotypic analysis of single cells using NGS.
Authors: Byrne-Steele Miranda , Pan Wenjing , Hou Xiaohong , Brown Brittany E. , Eisenhower Mary , Han Jian
Published: January 2023

Immune repertoire amplification of T and B cell receptors coupled to NGS provides detailed, sequence-level insight into the immune system. However, information related to the cognate pairing of alpha and beta chains is lost once RNA extraction is performed on the entire sample (or bulk sample). Here, we describe a very sensitive method that allows for the amplification of both human TCR alpha and beta chains including additional phenotypic markers from single cells in the same reaction well. We have reduced the RT-PCR chemistry as low as 1.5 μL, thus providing a cost-effective method to analyze single cells from standard FACs instruments. Plate processing procedures are also simplified since RT-PCR of all targets can be performed in the same plate. Under optimal conditions, the amplification success rate is greater than 90%, with successfully identified pairs as high as 75% of those amplified. During the first round of PCR, RT-PCR is performed with nested, multiplex primers covering both the alpha and beta locus (iPair) and 30 phenotypic markers (iPair+). Communal forward and reverse binding sites are included on the 5′ end of the inside primers. Included on the reverse primer is an in-line 6 nt barcode, which serves as a plate identifier so that multiple 96-well plates may be multiplexed in the same sequencing flow cell. After RT-PCR1, the first round PCR1 products are rescued using beads. A second PCR is performed with dual-indexed primers that complete the sequencing adaptors introduced during PCR1 and provide plate positional information for the sequenced products. An analysis tool has also been developed to help visualize pairing across a plate and to identify those pairs that would be likely candidates for further investigation.
Detection, phenotyping, and quantification of antigen-specific T cells using a peptide-MHC dodecamer
Authors: Huang Jun , Davis Mark M.
Published: January 2023

Here we report a peptide-MHC (pMHC) dodecamer as a “next generation” technology that is a significantly more sensitive and versatile alternative to pMHC tetramers for the detection, isolation, and phenotypic analysis of antigen-specific T cells. In particular, dodecamers are able to detect two- to fivefold more antigen-specific T cells in both human and murine CD4+ and CD8+ αβ T-cell compartments compared with the equivalent tetramers. The low-affinity, tetramer-negative, dodecamer-positive T cells showed comparable effector cytokine responses as those of high-affinity, tetramer-positive T cells. Dodecamers are able to detect early stage CD4+CD8+ double-positive thymocytes on which T-cell receptors are 10- to 30-fold less dense than mature T cells. Dodecamers also show utility in the analysis of γδ T cells and in cytometry by time-of-flight applications. This construct has a simple structure with a central scaffold protein linked to four streptavidin molecules, each having three pMHC ligands or other molecules. The dodecamer is straightforward and inexpensive to produce and is compatible with current tetramer technology and commercially available streptavidin conjugates.
The potential application of ctDNA for improved cancer management, treatment and diagnosis within the Pacific
Authors: Sika-Paotonu Dianne , Guilford Parry
Published: January 2023

The burden of cancer among Pacific peoples living in New Zealand and in the Pacific remains a significant health concern. Given the health inequalities that exist for Pacific peoples and the global burden of cancer, it remains a priority to consider the medical situation for cancer patients in the Pacific. It is acknowledged that within resource constrained environments, the diagnosis, treatment and management of cancer patients is fraught with unique challenges and difficulties. A novel cancer detection method known as circulating tumour DNA (ctDNA) monitors small fragments of tumour DNA found in the blood circulation. These fragments can be isolated and quantified to obtain information providing data concerning cancer progression and treatment response. It is anticipated this simple method of detecting and monitoring cancer will enhance the precision of cancer care for patients, and will eventuate into a particularly useful tool within resource constrained environments such as the Pacific. The purpose of this work was to identify the diagnostic, treatment and pharmacologic options currently available for cancer patient within the main and rural Hospitals in Tonga and to explore the potential utility of ctDNA to support the management, treatment and diagnosis of cancer patients. Cancer diagnosis, treatment and management pathways were available in Tonga but options were limited. The potential diagnostic utility of the ctDNA technology was an element of intense interest as cancer presentation is typically late with medical, nursing and support staff operating within resource constrained environments. The potential application of ctDNA for improved support of cancer patients in Tonga remains a highly appealing possibility.
Interferon epsilon protects primary macrophages against HIV infection
Authors: Chang Theresa Li-Yun , Tasker Carley , Subbian Selvakumar , Gao Pan , Couret Jennifer , Levin Carly , Ghanny Saleena , Soteropoulos Patricia , Zhao Xilin , Landau Nathaniel , Lu Wuyuan
Published: January 2023

Interferon epsilon (IFNe) is a unique type I IFN that is not induced by pattern-recognition response elements. IFNɛ is constitutively expressed in mucosal tissues including the female genital mucosa. Although the direct anti-viral activity of IFNe was thought to be weak compared to IFNa, IFNe controls Chlamydia muridarum and herpes simplex virus 2 in mice, possibly through modulation of immune response. We show here that IFNe induces an antiviral state in human macrophages that blocks HIV-1 replication. IFNe had little or no protective effect in activated CD4+ T cells or transformed cell lines unless activated CD4+ T cells were infected with replication competent HIV-1 at a low MOI. The block to HIV infection of macrophages was maximal after 24 h of treatment and was reversible. IFNe acted on early stages of the HIV life cycle including viral entry, reverse transcription and nuclear import. The protection did not appear to operate through known type I IFN-induced HIV host restriction factors such as APOBEC3A and SAMHD1. IFNe-stimulated immune mediators and pathways had the signature of type I IFNs but were distinct from IFNα2. IFNe induced significant phagocytosis and reactive oxygen species, which contributed to the block to HIV replication in macrophages. These findings indicate that IFNe induces an antiviral state in macrophages that is mediated by different factors than those induced by IFNa2. Understanding the mechanism of IFNe-mediated HIV inhibition through immune modulation has implications for prevention.
Transcriptome analysis reveals distinct immune response profiles in HBeAg+ and HBeAg− HBV infection and in HBV/HDV co-infection
Authors: Park Heiyoung , Heller Theo , Rehermann Barbara
Published: January 2023

In chronic hepatitis B virus (HBV) infection, HBeAg positivity is associated with an increase in liver inflammation and progression to liver cirrhosis and cancer. Super-infection with hepatitis D virus (HDV) results in more severe liver disease. The causative factors are unknown. To understand the unique immunopathogenesis of HBeAg+ chronic HBV infection and chronic HBV/HDV co-infection, we performed a comparative transcriptome analysis using peripheral blood mononuclear cells of 12 HBV infected patients (6 HBeAg+ vs. 6 HBeAg−), 10 HBV/HDV co-infected patients, and 12 uninfected controls using NanoString human immunology V2 panel.

About half of the differentially expressed genes in HBeAg+ versus HBeAg− HBV patients were interferon-stimulated genes (ISGs), and 83% of these ISGs (5/6) were upregulated. Ingenuity Pathway Analysis identified IFNA2 and IFNL1 as the most activated upstream regulators associated with HBeAg+ status.

In contrast, only 7 out of 31 differentially expressed genes in chronic HBV/HDV co-infection compared to chronic HBV infection were ISGs, which were either upregulated or downregulated, indicating that IFN-signature is marginally involved in the pathogenesis of chronic HBV/HDV co-infection. Instead, Ingenuity Pathway Analysis identified CD40LG and IL18 as the most activated upstream regulators, and IRF activation by cytosolic pattern receptors and B cell receptor signaling as the top canonical pathways associated with chronic HBV/HDV co-infection.

Our data suggest that IFN signaling may play a role in the enhanced liver disease found in chronic HBeAg+ HBV infection, and that CD40LG and IL18 signaling may contribute to unique immunopathogenesis of chronic HDV/HBV co-infection.
Fecal microbiota transplantation provokes systemic host immune changes that drive the clearance of lethal disseminated pathogens
Authors: Kim Sangman Michael , DeFazio Jennifer , Hyoju Sanjiv , Krezalek Monika , Khodarev Nikolai , Sangwan Naseer , Gilbert Jack , Zaborina Olga , Jabri Bana , Alverdy John
Published: January 2023

After several iterations across medical disciplines, fecal microbiota transplantation (FMT) is now widely and effectively used to treat recurrent C. difficile infection. FMT normalizes the composition of the gut microbiota and is widely thought to mediate its protective effects by displacing pathogens in the context of the gut.

We have previously shown that the microbiome of patients with severe sepsis consists of a low-diversity pathobiome dominated by multi-drug resistant pathogens. To test the hypothesis that treatment with FMT would protect against sepsis, we employed a gut-derived sepsis model that involves inoculation of a pathogen community (PC) into the gut of surgically-stressed mice. Septic mice receiving FMT derived from healthy mice via enema exhibited a dramatic increase in survival accompanied by restoration of gut microbial diversity. Intriguingly, we observed that treatment with FMT led to alterations to the host transcriptome in extra-intestinal tissues.

Given the systemic effects of FMT administration, we next investigated whether FMT could influence survival in a model in which the PC was acutely disseminated via i.p. injection. Remarkably, administration of FMT in the gut markedly increased survival and drove the systemic clearance of pathogens. This suggests that the effects of FMT are not merely mediated by gut-confined colonization resistance, but also through the modulation of systemic host physiology in a way that is detrimental to pathogens. Comparative transcriptomics suggested that in both models the FMT rescue effect depends on the upregulation of the IRF3/Type I IFN signaling pathways, and using knockout mice we demonstrated that IRF3 is required for FMT-mediated protection and pathogen clearance.
Protein immunization induces T follicular helper memory cells and uncommitted Th0- like memory cells
Authors: Hale J. Scott , Harrison-Chau Malia , Novis Camille Leite
Published: January 2023

We have previously demonstrated that viral infection generates lineage-committed T helper 1 (Th1) and T follicular helper (Tfh) memory cells that preferentially recall their lineage-specific functions following secondary reencounter with virus. In this study, we analyzed antigen-specific T helper cell differentiation and memory differentiation following glycoprotein in adjuvant immunization and compared it to differentiation induced by acute LCMV infection. Similar to viral infection, protein immunization induced CXCR5+ memory Tfh cells that rapidly recalled Tfh cell function following reactivation, while differing in their expression of certain transcription factors such as Tbet. Protein immunization also induced CXCR5- non-Tfh effector and memory cells. Surprisingly, these non-Tfh memory cells had undergone memory differentiation, but had not undergone polarization toward any particular T helper cell lineage. Upon challenge with virus, these Th0-like memory cells were able to generate Th1 effector cells. Together, these data demonstrate that protein-immunization generates Tfh memory cells, in addition to Th0-like memory cells that are relatively undifferentiated toward the T helper cell lineages.
A 2nd generation antiviral T cell product for sustained immunological regeneration
Authors: Amini Leila , Schmueck-Henneresse Michael , Volk Hans-Dieter , Reinke Petra
Published: January 2023

Reactivation of persisting viruses is one major complication in immunosuppressed patients. Despite the availability of potent antiviral drugs for CMV infection/reactivation, sufficient T cell response is required to control the infection sustainably, particularly in case of drug resistance development. In these cases, novel treatment strategies such as adoptive T cell therapy are indispensable.

We developed an autologous approach of antiviral T cell therapy which efficiently reduced viral load and symptoms, however, CMV disease relapsed in about half of patients. Back-to-bench, we could figure out that the relapses were presumably due to the biased composition of this 1st generation T cell product which almost exclusively consisted of short-lived effector memory T cells (TEM). Therefore, a 2nd generation protocol was developed which enriches for more long-lived and highly proliferative central memory T cells (TCM) which showed better engraftment and survival in animal models.

We characterized this 2nd generation T cell product employing different cytokine regiments in long-term culture with regard to stability, expansion, specific killing abilities, metabolic properties and could show its superiority with regard to cell survival and resistance to Fas-induced apoptosis.

Furthermore, we showed that TCM enrichment mainly resulted from preferred expansion and inhibited differentiation of TCM from the starting population, but partially also from reprogramming cells from “later” memory subsets, i.e. TEM and RA expressing effector T cells, into the TCM phenotype.

Finally, we demonstrated the applicability of this protocol to blood samples from kidney transplant patients who were undergoing immunosuppressive treatment.
Title: Human mucosal-associated invariant T (MAIT) cells in inflamed tissues
Authors: Berkson Julia D , Slicter Chloe , Dutta Mukta , Oberbillig Haley , Prlic Martin
Published: January 2023

Mucosal-associated invariant T (MAIT) cells recognize bacterial metabolites via a semi-invariant T cell receptor (TCR) presented by MHC-like molecule MR1. Once activated, MAIT cells are a potent effector population with cytotoxic and pro-inflammatory properties. We recently reported that a TCR signal is not sufficient to elicit effector function, instead pro-inflammatory cytokines in addition to a TCR signal are required for sustained MAIT cell effector functions. We proposed that this requirement allows MAIT cells to distinguish between commensal (TCR signal only) and pathogenic bacteria (TCR signal + inflammation). We next wanted to address how MAIT cells function within an inflamed tissue environment. Specifically, if inflammation resulted in increased MAIT effector function or an exhausted phenotype because of the sustained stimuli. We obtained gingival tissue and matched blood from donors undergoing periodontic surgery for various inflammation-related diseases. MAIT cells within inflamed mucosa showed increased effector function directly ex vivo compared to blood including expression of granzyme B and TNFa. RNAseq analysis of MAIT cells from inflamed mucosal tissue revealed a distinct transcriptional signature compared to MAIT cells in the blood. We observed a significant upregulation of chemotaxis and activation genes as well as inhibitory genes from multiple immune-modulatory pathways. Our data suggest that MAIT cells in inflamed tissues can maintain effector function, but also express immunomodulatory genes that presumably serve to avoid unwanted tissue damage.
High mobility group box 1 (HMGB1)-RAGE interaction mediates ectopic Intestinal inflammation in NAFLD.
Authors: CHANDRASHEKARAN VARUN , SETH RATANESH K , DATTAROY DIPTADIP , ALHASSON FIRAS , ZIELONKA JACEK , CARSON JAMES , BERGER FRANKLIN , KALYANARAMAN BALARAMAN , Diehl Anna Mae , Chatterjee Saurabh
Published: January 2023

Recent clinical studies found a strong association of colonic inflammation with NAFLD yet the mechanisms remain largely unknown. The present study identifies HMGB1 as a key mediator of intestinal inflammation in NAFLD and outlines a detailed redox signaling mechanism for such a pathway. NAFLD mice showed liver damage and release of higher HMGB1 in systemic circulation, increased intestinal tyrosine nitration that was dependent on NADPH oxidase. Intestines from NAFLD mice showed higher TLR4 activation and proinflammatory cytokine release, an outcome strongly dependent on the existence of NAFLD pathology and NADPH oxidase. In vitro experimentation using IE6 cells showed the HMGB1 activation of Toll like receptor (TLR-4) was both NADPH oxidase and peroxynitrite dependent with the latter being formed from the activation of NADPH oxidase. Proinflammatory cytokine production was significantly blocked by the specific peroxynitrite scavenger Phenyl Boronic Acid (FBA), AKT inhibitor (LY294002) and NADPH oxidase inhibitor Apocynin suggesting NADPH oxidase-dependent peroxynitrite as a key mediator in TLR-4 activation and cytokine release via an AKT dependent pathway. Studies to ascertain the mechanism of HMGB1-mediatied NADPH oxidase activation showed a distinct role of RAGE as the use of inhibitors targeted against RAGE prevented NADPH oxidase activation, peroxynitrite formation, TLR4 activation and finally cytokine release. Thus in conclusion, the present study identifies a novel HMGB1 mediated inflammatory pathway that is RAGE and redox signaling dependent and helps promote ectopic intestinal inflammation in NAFLD.
Silencing Mutated Cxcr4 in Mouse Hematopoietic Stem Cells as a Genetic Cure Strategy Obviating Bone Marrow Conditioning in WHIM Syndrome
Authors: Gao Jiliang , Yim Erin , Siwicki Marie , McDermott David H , Murphy Philip M
Published: January 2023

Genetic cure of diseases caused by autosomal dominant (AD) gain-of-function (GoF) mutations could require full correction of the disease allele; however, selective inactivation of the disease allele might suffice or even be superior. To test the relative efficacy of these two strategies in WHIM syndrome, a rare combined primary immunodeficiency disease caused by AD GoF mutations in chemokine receptor CXCR4 leading to impaired release of mature leukocytes from bone marrow, we have performed competitive bone marrow transplantation experiments with Cxcr4+/+, Cxcr4+/o and Cxcr4+/S338X (WHIM model) mice. Here we show a whole bone marrow donor genotype rank order of Cxcr4+/o >Cxcr4+/+> Cxcr4+/S338X for repopulating the blood of lethally irradiated recipient mice. Importantly, the strong competitive advantage of transplanted Cxcr4 haploinsufficient bone marrow cells is durable, intrinsic to hematopoietic stem cells (HSCs) and evident in the blood at the level of cell frequency and total cell numbers in both the myeloid and lymphoid lineages in both irradiated and unconditioned congenic recipients. Mechanisms included enhanced engraftment of hyperproliferative Cxcr4+/o HSCs in irradiated recipients and enhanced release of mature Cxcr4+/o leukocytes to the blood in unconditioned recipients. These results provide proof of principle that inactivation of the CXCR4 disease allele in HSCs may be a safe, effective and superior genetic cure strategy able to obviate toxic bone marrow conditioning in patients with WHIM syndrome.
Identification of lysophosphatidylserine receptor P2Y10 as a novel G-protein coupled receptor mediating eosinophil degranulation
Authors: Chung Il Yup , Jeong Kim Hye , Mi Hwang Sae
Published: January 2023

P2Y10, along with GPR34 and GPR174, is a G protein-coupled receptor that is activated by lysophosphatidylserine (LysoPS), and its expression and function have not yet been described. We have previously shown that P2Y10 is one of the highly upregulated genes at the late differentiation stage during in vitro eosinophilopoiesis. In the present study, we examine the expression and functions of P2Y10 in human eosinophils. P2Y10 is increasingly expressed, as cord blood (CB) CD34+ cells differentiate toward eosinophils. Analysis of its expression in peripheral blood (PB) leukocytes shows that P2Y10 is the most highly expressed in the granulocytes among the major leukocytes in numbers, including lymphocytes, monocytes, and granulocytes. Importantly, PB eosinophils express this receptor much more strongly than PB neutrophils. In contrast, GPR34 and GPR174 are negligible on PB eosinophils, while being comparable to P2Y10 in PB neutrophils. LysoPS activates CB eosinophils to induce a robust ERK phosphorylation as profoundly as IL-5. LysoPS is able to trigger degranulation of eosinophil cationic protein (ECP) in PB eosinophils, and its capability of degranulation is significantly blocked by inhibitors of signaling or receptor molecules including ERK, TACE, and EGFR. Taken together, our data suggest that LysoPS provokes a proinflammatory functions of eosinophils likely through P2Y10, proposing P2Y10 as a potential therapeutic target to control eosinophil-associated diseases.
Identification of Interleukin 40, a novel B cell-associated cytokine.
Authors: Catalan-Dibene Jovani , Vazquez Monica Ivonne , Luu Van Phi , Nuccio Sean-Paul , Maravillas-Montero Jose Luis , Villalta Sergio Armando , Ushach Irina , White Clayton , Pone Egest , Raffatellu Manuela , Hernandez Marcela , Zlotnik Albert
Published: January 2023

We have identified a novel mouse and human cytokine. IL-40 is normally produced in the bone marrow and fetal liver, and by activated B cells. Its sequence predicts a small (~27kDa) secreted protein unrelated to other cytokine gene families, that we have called Interleukin 40 (IL-40). IL40 is only present in mammalian genomes, suggesting a role in mammalian immune responses. Accordingly, IL-40 expression is induced in the mammary gland upon the onset of lactation, and an IL-40−/− mouse exhibits reduced levels of IgA in the serum, gut, feces, and milk. Furthermore, the IL-40−/− mouse has smaller and fewer Peyer’s patches and IgA secreting cells. The gut microbiome of IL-40−/− mice also exhibits altered composition, reflecting the reduced levels of IgA in the gut. We have also determined that B cell precursor populations are altered in the IL-40−/− mouse. Taken together, these observations indicate that IL-40 represents a novel B cell associated cytokine, that plays an important role in the development of humoral immune responses. IL-40 is also expressed by human activated B cells and by several human B cell lymphomas. The latter observation suggests that it may play a role in the pathogenesis of these diseases.
The impact of IL-27 on human T cells is altered in multiple sclerosis patients
Authors: Clénet Marie-Laure , Beauseigle Diane , Devergne Odile , Duquette Pierre , Prat Alexandre , Arbour Nathalie
Published: January 2023

IL-27 exhibits pro- and anti-inflammatory properties. This cytokine dampens the severity of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). Whether IL-27 plays a role in pathobiology of the human disease is still unresolved. We recently showed that both IL-27 and IL-27Receptor (IL-27R composed of IL-27Rα and gp130 chains) are elevated in MS brains compared to controls. We reported expression of IL-27R by brain-infiltrating CD4 and CD8 T cells in MS tissues. To determine whether IL-27 contributes to altered immune responses in MS, we investigated the impact of this cytokine on peripheral blood T cells obtained from untreated MS patients and age/sex-matched healthy donors. Surface IL-27R is present on lower proportions of effector memory (CCR7−) CD4 and CD8 T cells compared to naïve (CCR7+CD45RA+) and central memory (CCR7+CD45RO+) counterparts in all donors. However, reduced proportions of all CD4 T cell subsets but enhanced percentages of CD8 T cell subsets express IL-27R in MS patients compared to controls. IL-27 triggers rapid phosphorylation of STAT1 and STAT3 in all T cell subsets. The percentage and intensity of pSTAT1 detection in all T cell subsets were similar in both donor groups. In contrast, increased proportions of all CD4 and CD8 T cell subsets express pSTAT3 in response to IL-27 compared to cells from controls. Finally, elevated amounts of both IL-27 and its recently identified natural antagonist (soluble) IL-27Rα are present in serum from MS patients compared to controls. Thus, our results reveal that IL-27 has an altered impact on T cells from MS patients which could contribute to aberrant immune responses associated with this inflammatory/autoimmune disease of the central nervous system.
Modulation of Inflammation Induced by Recombinant MOMP of Chlamydia in Mouse Macrophages by IL-10 is Mediated by Skewing M1 to an M2 Polarizing Macrophage Phenotype
Authors: Duncan Skyla A. , Sahu Rajnish , Martin David , Singh Shree R , Dennis Vida
Published: January 2023

The major outer membrane protein (MOMP) of Chlamydia trachomatis (CT) is a very desirable vaccine candidate against CT because it stimulates robust T- and B-cell protective responses. Conversely, we showed that MOMP contributes to CT inflammation by stimulating macrophages to secrete pro-inflammatory cytokines that play pivotal roles in the pathogenesis of chlamydial diseases, and whose secretion levels are modulated by IL-10. We also showed that MOMP induced marked expression of the Socs3 gene in macrophages, correlating with its induction of pro-inflammatory cytokines, and its triggering of an M1 macrophage phenotype. Our goal in this study is to understand the inflammatory nature of MOMP by focusing on key chemokines (CCL5 and CXCL10), NO and Nod1/2 receptors, which are implicated in CT inflammation. We also evaluated their inhibition by IL-10 and its effect on M1 and M2 macrophage phenotypes. Mouse J774 macrophages were stimulated with recombinant MOMP (0.01–10 ug/mL) with/without exogenously added IL-10 (10 ng/mL); supernatants and RNA were collected for cytokine ELISAs, NO production and TaqMan gene expression analyses. Our results showed that rMOMP induced expression of CCL5, CXCL10, NO and Nod1/2 receptors that recognize and activate key inflammatory pathways, which were all modulated by IL-10. Characterization of macrophages revealed that rMOMP increased Nos2 expression, an M1 phenotypic marker, which was modulated by IL-10, suggesting skewing of the M1 phenotype whereas IL-10 upregulated M2 markers including, Arg 1 (and its isoform Arg 2) and Mrc1. Overall, our data helps in understanding the inflammatory nature of MOMP and the potential role played by M1 and M2 phenotypes in mediating IL-10 regulation of CT inflammation.
GM-CSF upregulates IL-15 expression on specific human myeloid cells.
Authors: Farzam-kia Negar , Duquette Pierre , Prat Alexandre , Arbour Nathalie
Published: January 2023

Although the salience of the immune system in multiple sclerosis (MS) pathology has been established, the mechanisms and mediators involved are incompletely characterized. We have identified IL-15, which aids in the development, activation, and survival of immune cells, as a relevant contributor to MS neuropathology. We previously showed that greater proportions of myeloid cells, including peripheral monocytes and brain macrophages/microglia, from MS patients express IL-15 versus controls. Thus, we aim to identify the factors that contribute to these elevated IL-15 levels in the context of MS. Primary human monocytes from MS patients and age/sex matched healthy donors are treated with M-CSF to trigger differentiation into macrophages (considered M0 macrophages) prior to being polarized into M1 or M2 macrophages. While a small proportion of M0 macrophages express IL-15, M2 polarization significantly enhances the percentage of IL-15+ macrophages. In contrast, M1 polarization does not affect IL-15 levels although it significantly increases MHCI and CD80 expression. GM-CSF is produced by pathogenic encephalitogenic T cells; monocytes and macrophages express the corresponding receptor (GM-CSF receptor). GM-CSF induces a major increase in the proportion of IL-15+ M0 and M2 macrophages from both donor groups. In contrast, GM-CSF does not modify IL-15 expression by monocytes although it induces STAT5 phosphorylation. In conclusion, GM-CSF is a potent trigger for IL-15 expression on M0/M2 macrophages but not monocytes. Moreover, M2 polarization of macrophages increases IL-15 expression. Identification of IL-15 triggers and further characterization of IL-15+ immune cells may provide novel targets for therapies for MS patients.
The CD8+ T-cell receptor (TCR) repertoire in extremely preterm neonates is highly shared and convergent
Authors: Carey Alison J , Hope Jennifer L , Fike Adam J , Mueller Yvonne M , Nguyen Linda T , Kumova Ogan K , Van Zessen David BH , van der Burg Mirjam , Katsikis Peter D
Published: January 2023

Neonates have an immunological immaturity which persists from fetal life into the newborn period and can result in a poorly protective response to intracellular pathogens. We have previously shown a less complex TCR repertoire may be responsible for reduced CD8+ T cell immunity in a neonatal murine model of influenza virus infection. Therefore, we sought to determine if there were similar differences in the TCR repertoire across the human life span. High-throughput sequencing of the TCRβ CDR3 regions was performed in sorted, naïve CD8+ T cells from extremely preterm (23–27 weeks gestation), term (39–40 weeks gestation), children aged 16 months to 4 years old, and adults aged 25–50 years (n=5 in each group). There were differences in the CDR3 length across the life span, with preterm infants having a shorter CDR3 length, which was due to fewer N1 and N2 additions (p<0.01) than their term counterparts, which inherently decreases junctional diversity. There was no increase in trimming at the 5′ and 3′ ends of the VDJ genes across the lifespan. Preterm neonates had a striking number of shared clonotypes (8% of total clonotypes) versus their term counterparts (3%) and adults (1%). Convergent recombination describes the phenomenon that identical TCR specificities at the amino acid level can be obtained through different nucleotide sequences. Upon analysis of the most highly shared clones (common to at least 4 samples/age group), the preterm neonate had 12 nucleotide rearrangements per highly shared amino acid sequence, compared to 6 rearrangements at term and 5 in the adult. Our analysis demonstrates the prevalence of shared, convergent clonotypes within the preterm TCR repertoire suggestive of an antigen-driven shaping of the TCR repertoire in utero.
Age-associated loss of lamin-B1 in thymic epithelial cells disrupts the thymic microenvironment for thymopoiesis and leads to thymic involution
Authors: Yue Sibiao
Published: January 2023

Lamins are abundant type V intermediate filaments important for the structure and function of the nucleus. By studying the Drosophila immune organ, the fat body, recent work in our lab has uncovered a mechanism by which lamin-B loss in the fat body upon aging induces age-associated systemic inflammation and gut hyperplasia, suggesting that lamin-B loss triggers a phenomenon known as immunosenescence. A role of B-type lamins in mammalian immune aging, however, remains unexplored. Here, we report an age-related reduction of lamin-B1 inthymic epithelial cells (TECs) in the thymus, the primary immune organ for T-cell generation. We further demonstrate that genetic ablation of lmnb1 in TECs causes remarkably similar phenotypes as these observed in aged thymus. Furthermore, RNA-seq analyses reveal that depletion of lmnb1 accelerates age-related transcriptional changes in TECs, suggesting that loss of lamin-B1 in TECs upon aging may lead to global transcriptome alterations and trigger a degenerative cascade in the thymus. This work provides new insights into the cause and consequence of immunosenescence during mammalian immune aging.
Psmb11 molds the transcriptome of cortical TECs and CD8 thymocytes
Authors: Apavaloaei Anca , Rouette Alexandre , Brochu Sylvie , Perreault Claude
Published: January 2023

Psmb11 is a catalytic subunit of the thymoproteasome, expressed exclusively in cTECs (cortical thymic epithelial cells). It has been shown that Psmb11-mediated positive selection is pivotal for the generation of an immunocompetent T-cell repertoire. We found that lack of Psmb11 results in a suite of gene expression changes in cTECs and not in mTECs, about 200 genes with enrichment in cell-cell interaction and antigen processing and presentation. One yet unsolved question is why the cellularity of CD8+ single-positive (SP8) but not SP4 thymocytes decreases strongly as a consequence of Psmb11-deficiency. Our transcriptomic analyses of pure SM, M1 and M2 subsets of developing SP4 and SP8 T cells show that the maturation of SP4 cells is essentially independent of Psmb11. However SP8 thymocytes present a dysregulated development with impaired migration, survival and lineage commitment in Psmb11 knock-out mice, also detected at the protein level by flow-cytometry. Thymocytes present decreased surface levels of CD127 on SP8 and slightly increased CD5 on all subsets. This indicates a stronger activation of the developing T cells, which alone cannot explain the impact on the SP8 cellularity. We have identified a novel role of Psmb11 in modulating, directly or indirectly, the expression of genes in cTECs, which is necessary to guide and complete the positive selection of SP8 thymocytes.
CCR7 modulates the generation of thymic regulatory T cells by altering the composition of the thymic dendritic cell compartment
Authors: Ehrlich Lauren Ilyse Richie , Hu Zicheng , Li Yu , van Nieuwenhuijze Annemarie , Kessler Rachel A , Liston Adrian
Published: January 2023

Upon recognition of auto-antigens, thymocytes are negatively selected or diverted to a regulatory T cell (Treg) fate. Both dendritic cells (DC) and medullary thymic epithelial cells (mTEC) present antigens to tolerize thymocytes. CCR7 guides thymocytes into the medulla, promoting encounters with medullary DC and mTEC. Thus, in the absence of CCR7 negative selection of auto-reactive thymocytes is impaired and autoimmunity ensues. Here we describe an unanticipated, non-thymocyte intrinsic contribution of CCR7 to intrathymic Treg generation. We find that Ccr7−/− mice have increased Treg cellularity, due to a hematopoietic, but non-T cell autonomous function of CCR7. In addition to thymocytes, thymic DC also express CCR7, which promotes survival of Sirpα−DC. Without CCR7, Sirpα−DC are preferentially lost, altering the composition of the conventional thymic DC compartment to favor intrathymic Treg generation. CCR7 deficiency results in enhanced production of thymic Treg at the neonatal stage and in lymphodeficient adults, when Treg generation is most critical for the establishment of self-tolerance. Together these results reveal a more complex function for CCR7 in thymic tolerance induction, in which CCR7 not only promotes negative selection through its effect on thymocyte localization, but also governs intrathymic Treg generation via non-thymocyte intrinsic mechanisms.
AIF1 expression regulates differentiation of dendritic cells from hematopoietic stem cell multipotent progenitor subsets and regulates antigen presentation capacity
Authors: Elizondo Diana Margarita , Andargie Temesgen Ewunetu , Lee Clarence Matthews , Anderson Winston Anthony , Lipscomb Michael Wheeler
Published: January 2023

Dendritic cells (DC) are indispensable in orchestrating adaptive immunity, serving as the principal messengers between innate and adaptive arms. DC subsets are derived from CD34+ hematopoietic stem cells (HSC). DC development progress through differentiation from HSC multipotent progenitors towards common myeloid progenitors. However, additional proteins involved in this selective differentiation process remain to be identified. For these studies, use of transcriptomic profiling and RNA interference (RNAi) approaches have now identified that Allograft Inflammatory Factor-1 (AIF1) directly contributes to DC differentiation and immunomodulatory processes. HSC multipotent progenitors, identified as CD117+Lin-CD34+SCA1+, were found to not express AIF1. However, CD117+Lin-CD34+SCA1- and CD117+Lin-CD34-SCA1- subsets, known as the oligopotent progenitors, did express AIF1. Importantly, this was restricted to common myeloid progenitors, as the common lymphoid progenitors, defined as CD117+Lin-SCA1-IL-7Rα+ did not express AIF1. RNAi knockdown of AIF1 in sorted Lin-CD34−CD117+SCA1+ cells led to restrained differentiation in DC upon culturing with GM-CSF. Interestingly, knockdown of AIF1 in committed CD11c+ DC resulted in impaired antigen presentation to cognate CD4+ T cells in vitro, suggesting an important immunostimulatory role in committed myeloid lineages. Current studies are now adoptively transferring HSC knocked out for AIF1 into mice to assess in vivo DC differentiation ability and capacity to migrate and populate the splenic compartment. This is the first report showing the novel role of AIF1 in dendritic cell development and antigen presentation.
Ras, Erk, and PI3K signaling pathways in the central selection of B cells
Authors: Greaves Sarah A , Babolin Chiara , Torres Raul M , Pelanda Roberta
Published: January 2023

B cell selection mechanisms in the bone marrow control the nature of the primary B cell repertoire, generating diverse specificities while reducing autoreactivity. Our studies aim to characterize the mechanisms that drive the positive selection of immature B cells in the bone marrow. It has been shown that the B cell receptor (BCR) is able to signal in the absence of ligand (tonic signal) and this signal is required for B cell development. We hypothesize that alterations in tonic signaling could cause aberrant positive selection of autoreactive immature B cells. We have previously shown that low levels of active Ras, Erk, and Akt correlate with levels of BCR tonic signal and that constitutive activation of Ras allows for differentiation of autoreactive immature B cells in vitro. We are currently testing the individual contributions of the Erk and PI3K pathways in this selection process. By using retroviral transduction of primary bone marrow B cells in vitro, we show that Erk activation can overcome a lack of tonic signaling to induce differentiation of nonautoreactive or slightly autoreactive B cells. We have also found that the Dual Specificity Phosphatase 5 (DUSP5), a novel phosphatase, may play a role in Erk regulation and the selection of immature B cells. In order to study these pathways in vivo we generated mouse models in which constitutively active forms of MEK or PI3K are specifically expressed in autoreactive B cells to determine if activation of these molecules is sufficient to bypass central tolerance. Preliminary data suggests that in vivo activation of PI3K can drive development of autoreactive B cells and allow some escape to the periphery.
Foxn1-β5t transcriptional axis controls CD8+ T cell production in the thymus
Authors: Uddin Muhammad Myn , Ohigashi Izumi , Takahama Yousuke
Published: January 2023

The thymus is an organ that produces functionally competent T cells that protect us from pathogens and malignancies. Foxn1 is a transcription factor that is essential for thymus organogenesis; however, the direct target for Foxn1 to actuate thymic T cell production is unknown. Here we show that a Foxn1-binding cis-regulatory element promotes the transcription of β5t, which has an essential role in cortical thymic epithelial cells to induce positive selection of functionally competent CD8+ T cells. A point mutation in this genome element results in a defect in β5t expression and CD8+ T cell production in mice. The results reveal a Foxn1-β5t transcriptional axis that governs CD8+ T cell production in the thymus.
Stroma-free, serum-free differentiation and expansion of hematopoietic stem and progenitor cells to the T cell lineage
Authors: Tabatabaei-Zavareh Nooshin , Le Fevre Tim A , Man Alexander JY , Karas Evan A , Szilvassy Stephen J , Thomas Terry E , Eaves Allen C , Wognum Albertus W
Published: January 2023

T cells are produced by the differentiation of hematopoietic stem and progenitor cells (HSPCs) into Lin− CD34+ CD7+ progenitors that migrate from the bone marrow (BM) to the thymus where further T cell development takes place. The differentiation of HSPCs into T cells can be supported in vitro by using OP9 stromal cells that express a Notch ligand. We have developed a more simple stroma-free culture method that promotes the expansion and differentiation of T cell progenitors from purified CD34+ HSPCs and purified multipotent lymphoid progenitors (MLPs) from cord blood (CB) or BM. CD34+ CB cells isolated using EasySep magnetic separation or CD34+ CD38− CD90− CD45RA+ MLPs further purified by cell sorting were seeded into culture plates coated with an attachment substrate containing Notch ligand and cultured in serum-free StemSpan SFEM II medium in the presence of a cytokine supplement containing SCF, TPO, Flt3L and IL-7. After 3 weeks, CD7+ CD5+ proT cells were detected at a frequency of 84% (range: 63–97%, n=26) with a proT cell yield of 2,130 (142–5,970, n=26) per initial CD34+ cell and 28,110 (4,720–63,930, n=4) per MLP initially seeded. In these cultures 28% (12–43%, n=12) of CD7+ cells also expressed CD1a, indicating further differentiation into preT cells. The pro and preT cells could mature into CD4+ CD8+ double positive T cells (38%; 24–64%, n=6) and TCRαβ+ CD3+ T cells (12%; 5–23%) over two weeks of continued culture in the same medium and in the presence of OP9-DL1 stromal cells. These results demonstrate that HSPCs can expand and differentiate into pro and preT cells under stroma-free and serum-free culture conditions. This novel culture system may benefit research and development of immunotherapies where large numbers of T cells are required.
Influenza virus induces mast cell death independent of mast cell activation
Authors: Temple Rachel Mary , Obar Joshua J
Published: January 2023

Influenza A virus (IAV) infects over a million individuals each year, resulting in significant morbidity, and during pandemic years, significant mortality. Severe IAV infection is characterized by a dysregulated cytokine response, termed a ‘cytokine storm’. Recent evidence indicates mast cells can play a central role in initiating this cytokine storm, though the long-term effects of IAV infection on mast cells is poorly understood. To this end, we infected bone marrow cultured mast cells with IAV at a low multiplicity of infection, allowing us to track IAV infection in mast cell cultures over time. Within hours mast cells express viral proteins, but fail to release appreciable levels of infectious viral particles throughout the course of infection. Despite the abortive nature of this infection, it is highly cytotoxic, with significant cell death occurring between 12–48 hours post infection. This cell death is the result of simultaneous activation of apoptosis and necroptosis pathways. Remarkably, while UV-inactivated virus activates mast cells, it does not induce cell death. Conversely, infection with a neuraminidase mutant is cytotoxic despite the fact this mutant fails to activate mast cells. These data suggest the induction of programmed cell death is dependent on viral replication, but independent of mast cell activation. In support of this hypothesis, the RIG-I and interferon signaling pathways are dispensable for the induction of cell death, as mast cells derived from MAVS−/−, AG129 (IFNAR1/IFNGR DKO), and STAT1−/− mice die at rates comparable to WT cells. Together, these data indicate mast cells detect IAV infection using multiple innate sensors, with each mode of detection resulting in the activation of a distinct response.
Assessment of lymph node stromal cells as an underlying factor in age related immune impairment to influenza infection
Authors: Masters April , Hall Alexxus L. , Haynes Laura
Published: January 2023

It is well established that aging negatively impacts immunity, resulting in inefficient responses to vaccinations and infections, like influenza. Fibroblastic reticular cells (FRCs) are the major stromal cell subset in lymph nodes and play an intricate role in the orchestration and control of adaptive immune responses. Although stromal cells have a major impact on immune responses, the impact of aging on LN stromal cells has yet to be determined. We sought to determine how aging impacts FRC number, phenotype and morphology at steady state and after influenza infection. An enzymatic and mechanical digestion was used to make single cell suspensions of lymph node stromal cells for analysis. Flow cytometry was performed to quantify the number and frequency of stromal cells in lymph nodes from young (13 weeks) and aged (19 months) C57BL/6 mice. To analyze stromal cell morphology, lymph nodes from young and aged mice were cryopreserved, and then sectioned for confocal microscopy. Quantitative analysis of lymph node stromal cells by flow cytometry revealed that there are no significant differences in the number stromal cells in young and aged lymph nodes at steady state, but after influenza infection aged FRCs fail to expand in number. Reduced proliferation and activation of aged FRCs, but not increased death leads to the decreased expansion. Microscopic analysis revealed that young and aged LN T cell zone FRCs have similar morphology at steady state and after infection. These results are the first evidence that aging alters lymph node stromal cells ability to respond to challenge. These changes in aged FRCs may be an underlying cause behind the impaired immune response to influenza infection found in the elderly.