bluuebunny/crossref_metadata_2025 huggingface dataset:
MiR150 over-expression impairs iNKT cell development, maturation, and function
Authors: Wu Xiaojun , Zhou Li , Wang Jie , Li Guihua , Mi Qing-Sheng
Published: January 2023

CD1d-restricted invariant natural killer T (iNKT) cells play an important role in the regulation of diverse immune responses. MicroRNAs have been shown to be critically involved in iNKT cell development, maturation, and function, but the roles of specific miRNAs in it remain not completely understood. We had previously shown the dynamic upregulation of miR-150 during thymus iNKT cell maturation and identified the interrupted iNKT final stage maturation and elevated IFNγ production in miR-150KO mice. To determine the requirement of dynamic expressional changes of miR150 during iNKT cell development, we generated thymus-specific miR150 knock in mice (miR-150KI). MiR-150 over expression resulted in reduced overall iNKT cell number in both thymus and periphery, which could be related to increased apoptosis. MiR-150KI thymus iNKT cells showed the blockade of iNKT maturation at multiple maturation transition points. Splenic iNKT with miR-150KI displayed interrupted maturation and reduced cytokine production, including IFNγ, IL-4, and TNFα, upon in vitro stimulation. Furthermore, Bone marrow chimera study confirmed the effect of miR-150KI on iNKT cell development, maturation, and function are cell autonomous. Altogether, our results indicated that the tight control of miR150 expression is required in iNKT cell development and function. Detailed molecular mechanisms of miR-150 mediated iNKT cell regulation in different developmental stages and activation status are currently under investigation.
IDO1 activity in selected immune cells controls antibody responses
Authors: Scalisi Giulia , Gargaro Marco , Turco Antonella , Iorio Alfonso , Puccetti Paolo , Matino Davide , Fallarino Francesca
Published: January 2023

Indoleamine 2,3-dioxygenase 1 (IDO1) is an enzyme involved in the initial step of tryptophan degradation along the kynurenine pathway. It has immunosuppressive effects, linked to enzymic and non-enzymic regulatory functions. IDO1 is also crucial for sustaining the function of T regulatory cells (Treg), which are involved in the establishment of peripheral tolerance.

We recently found that defective IDO1 induction is associated to anti-FVIII antibody production in patients with severe Hemophilia A1. In hemophilic mice, CpG- rich oligodeoxynucleotides (CpG-ODN) administration induced IDO1 expression in dendritic cells (DCs) that control FVIII antibody response.

Recently we discovered that CpG-ODN selectively induced IDO1 in a subset of conventional DCs (cDCs). IDO1 was found to be highly expressed in gut CD11C+CD103+ DCs, which are required for establishment of oral tolerance2. Based on these data we analyzed the impact of IDO1 deficiency in controlling antibody responses, in two different experimental models (FVIII and ovalbumin, OVA, immunization). Interestingly, IDO1 deficiency resulted in a significant increase of FVIII-specific antibody production relative to wild type controls. Similarly, IDO KO mice showed a significant immunoglobulin production in a model of OVA-induced oral tolerance.

These data suggest the regulatory role of IDO1 in controlling antibody responses to self and non-self antigens.
Pam3CSK4 induces MMP-9 expression in human monocytic THP-1 cells
Authors: Alrashed Fatema , Kochumon Shihab , Sindhu Sardar , Ahmad Rasheed
Published: January 2023

Background

Matrix metalloproteinase (MMP)-9 is known to degrade the extracellular matrix and increased MMP-9 levels are related with the pathogenesis of many inflammatory conditions including obesity. Pam3CSK4 is a synthetic triacylated lipopeptide (LP) which is a potent activator of immune cells and induces cytokine production. However, it is unclear whether Pam3CSK4 is able to induce MMP-9 expression in monocytic cells. We, therefore, determined MMP-9 production by Pam3CSK4-treated THP-1 cells and also investigated the signal transduction pathway(s) involved.

Methods

MMP-9 expression was determined by real-time qPCR and ELISA and MMP-9 activity was assessed by zymography. THP-1 cells, THP1-XBlue™ cells, THP1-XBlue™-defMyD cells, anti-TLR2 mAb and selective pharmacological inhibitors were used to study signaling pathways involved. Phospho and total proteins were detected by western blotting.

Results

Pam3CSK4 induced MMP-9 expression (P<0.05) at both mRNA and protein levels in human monocytic THP-1 cells. Increased NF-κB/AP-1 activity was detected in Pam3CSK4-treated THP-1 cells and MMP-9 production in these cells was significantly suppressed by pre-treatment with anti-TLR2 neutralizing antibody or by inhibition of clathrin-dependent endocytosis. Also, MyD88−/− THP-1 cells did not express MMP-9 following treatment with Pam3CSK4. Inhibition of JNK, MEK/ERK, p38 MAPK and NF-κB significantly suppressed MMP-9 gene expression (P<0.05).

Conclusion

Pam3CSK4 induces MMP-9 production in THP-1 cells through the TLR-2/MyD88-dependent mechanism involving ERK, JNK, p38 MAPK and NF-κB/AP-1 activation.
Toll-Like Receptor (TLR)-2 Mediates Recruitment of Neutrophils During Influenza Infection in a Neonatal Murine Model
Authors: Kumova Ogan K , Nguyen Linda T , Fike Adam J , Carey Alison J
Published: January 2023

Infants less than six months of age are particularly susceptible to influenza infection due to their impaired cytotoxic T cell response. The neonate relies on innate immunity to contain infection without excessive inflammatory response, by using pattern recognition molecules, like collectins and TLRs. TLR2 has been identified as a bacterial recognition receptor. However, little is known about its role in viral recognition and associated pathogenesis, particularly in the neonate. To address this, 3-day old TLR-2−/− and control C57Bl/6 mice were infected with influenza type A virus strain PR/8/34 (H1N1) and tracked for survival. Bronchoalveolar lavage fluid (BALF) was collected and lungs were harvested at post infection day (PID) 1, 3, and 6. BALF was analyzed by flow cytometry for cellular infiltration in the airways. RNA was isolated and analyzed via real-time PCR for viral loads and various cytokines. There was a striking difference in survival between the TLR-2−/− (61%) and control neonates (25%) (p< 0.01). Surprisingly, TLR-2−/− mice had higher viral loads than controls (p < 0.05) on PID 1, which was coupled with lower neutrophil numbers in the BALF (p < 0.02). No significant differences were noted on PID 3 or 6 for neutrophil counts or viral loads. On PID 3, TLR-2−/− mice had more than a 2-fold lower transcription of CXCL-5, a key chemoattractant for neutrophils. Therefore, the resistance of neonatal TLR-2−/− mice to influenza infection was not due to heightened influenza clearance. Rather, there was impaired neutrophil recruitment, which potentially reduced pulmonary inflammation. Future studies using our neonatal murine model could elucidate the mechanism of TLR-2 and CXCL-5 mediated inflammation during neonatal viral infection.
Parenteral-Mucosal prime-boost with a TB subunit vaccine enhances early lung T cells against aerosol M. tuberculosis infection with transient improved protection.
Authors: Woodworth Joshua S , Christensen Dennis , Andersen Peter
Published: January 2023

Background

T cell mediated immunity is critical to control M.tuberculosis (Mtb) infection and tuberculosis (TB) disease. However, Mtb actively delays the adaptive T cell response after initial infection. Thus, a cornerstone of TB vaccine development is to overcome this delay and recruit T cells for direct contact with infected lung phagocytes to control early bacterial growth.

Given parenterally, the H56:CAF01 subunit vaccine results in an early CD4 T cell response in Mtb-infected lungs and significant reduction in CFU. Here, we investigate mucosal prime-boost delivery of H56:CAF01 to elicit a lung-localized/homing memory T cell population for further accelerated lung T cell recruitment and Mtb control.

Methods

CB6F1 mice vaccinated with H56 (Ag85B-ESAT6-Rv2660) adjuvanted with CAF01 (Th1/Th17-skewing liposomes) either 3× s.c., or 2× s.c. + i.n. boost(s) were rested to memory phase and then challenged with aerosol Mtb. Tissue-localized Mtb-specific T cells were enumerated by flow cytometry, and bacterial CFU determined. In vivo intravascular staining identified lung-localized from vasculature-localized T cells.

Results

Prime-boost with H56 resulted in a 10-fold increase in recruited/expanded Mtb-specific T cells in the lung parenchyma early after Mtb challenge over s.c.-only vaccination and a reduction in lung CFU at peak infection. However, no CFU difference was observed the chronic stage. Inhibition of S1P1 did not enhance the early protective effect, suggesting against local Trm as driving protection. Overall, the limited increased protection despite significant lung T cell enhancement suggests a constrained ability of vaccine T cells to control early Mtb replication.
Upregulation of innate signaling upon ligation of FcγRIIIa by immune complexes in Human CD4+ T-cells
Authors: Chauhan Anil K
Published: January 2023

Over the past two decades, role of Fc-receptor (FcR) signaling in CD4+ T-cell responses has been overlooked. Whether FcRs signaling in CD4+ T-cells contribute to tolerance breakdown and nucleic acid sensing remain an open question. We earlier reported that the FcγRIIIa cosignaling differentiates human naïve CD4+ T-cells in the absence of CD28 cosigning (JBC, 5128: 2016). We now show that the FcγRIIIa signaling upregulate the expression of nucleic acid sensing toll-like receptors (NA-TLRs) in naïve human CD4+ T-cells. TLR9 accumulate as a full length 120-kDa protein on the cell surface (revision in JI). TLR9 bound to CpG ODN 2006 and localized with FcγRIIIa on the cell surface. Subcellular location of the TLR9 discriminate between modified self-nucleic acid from encapsulated viral nucleic acids. FcγRIIIa signal relocated MyD88 and HMGB1 proteins underneath cell surface with TLR9 forming “myddosomes”. RNA-seq data showed upregulation of several key pathways, which are normally observed during innate immune responses. FcγRIIIa signal upregulated RNA transcripts encoding for IL-1α, IL-1β, IL-2, IL-17A, IL-22, IL-23, IL-31RA, and IL-34 cytokines. Transcripts from IFN subsets, IL-27, IFNι-2, IFNι-3, IFNω, IFNβ, IFNα and IFN inducible genes were upregulated. TLR9 signaling drives the production of IFN-γ, IL-17A, IL-21, ICOS and Bcl6 proteins. Statistically significant increase in the genes from cellular response to DNA damage; RNA-binding; and ERK-signaling was observed. lncRNAs populate exosomes and also regulate TLRs, which were unregulated by FcγRIIIa signal. Our data show that the FcγRIIIa drives innate signaling pathways often observed during inflammation, which may contribute to the naive CD4+ T-cell differentiation.
Functional plasticity of human Tc17 cells is dependent on cytokine milieu
Authors: WU MEI-YAO , YEN HUNG-RONG
Published: January 2023

Human Tc17 cells (IL-17-producing CD8+ T cells) have been identified, but little is known about their functional characteristics and mechanism of their plasticity. We aimed to optimize the differentiation of Tc17 cells and then investigate whether the reprograming of Tc17 to Tc1 cells can promote the anti-tumor cytotoxicity. In this study, we isolated CD8+ T cells from the peripheral blood of healthy donors to induce Tc17 differentiation. Human CD8+ T cells were stimulated with anti-CD3/anti-CD28 antibodies, TGF-β, IL-1β, IL-6, IL-23, IL-2, anti-IFN-γ, and anti-IL-4 antibodies. After 10 days in culture, Tc17 cells were about 10% IL-17+. Although Tc17 cells had upregulated anti-apoptotic proteins, the expression of cytotoxic molecules and lytic function of Tc17 cells were weaker than Tc1 cells. Interestingly, Tc17 cells could be converted to Tc1 cells under the stimulation of specific cytokine milieu, and their lytic function improved after conversion. Given these findings of significant enhancement of cytotoxicity, we suggest that reprogramming of human Tc17 cells can be used as an immunotherapeutic strategy against cancer.
Assessing metabolic demands in B cells
Authors: Mielenz Dirk , Urbanczyk Sophia , Stein Merle , Reimer Dorothea , Winkler Thomas , Jäck Hans-Martin
Published: January 2023

Assessing metabolism during early B cell development we show here that large pre B cells are superior in their metabolic activity but small pre B cells down-regulate glycolysis significantly, both ex vivo and in IL-7 cultures. The mitochondrial Ca2+ binding protein Swiprosin-2/EFhd1 (EFhd1) has been proposed to balance mitochondrial Ca2+ and the mitochondrial membrane potential. We show here that EFhd1 is expressed in pro B cells but becomes downregulated by surface expression of the pre BCR. Conversely, EFhd1 becomes up-regulated again in plasma cells. A transgenic mouse model, where EFhd1 cannot be downregulated by the pre BCR anymore (EFhd1tg), revealed that the mitochondrial metabolic regulator PGC-1a is upregulated by EFhd1. This lead to mitochondrial dysfunction in EFhd1tg pre B cells, to a disadvantage for EFhd1tg pro and pre B cells in mixed bone marrow chimeras and to an increased mitochondrial proton leak in EFhd1tg LPS blasts. In accordance, EFhd1 transfected into 293 cells is enriched in the mitochondrial fraction and co-localizes with mitochondria. On the other hand, CRISPR-Cas9 mediated knock-out of EFhd1 in the pro B cell line 38B9 as a surrogate for the pre BCR signal increased glycolysis, glycolytic capacity and reserve, providing a mechanism for increased metabolic activity in large pre B cells. Thus, proper regulation of EFhd1 is important to equilibrate mitochondrial and glycolytic metabolism during B cell differentiation. To gain more insight into metabolic demands and mechanisms during B cell development, activation and differentiation we generated EFhd1KO mice. First experiments with EFhd1KO mice revealed an important function for EFhd1 during B cell development, differentiation and function.
Unique properties of polarized M1 and M2 macrophages from 2′-5′ Oligoadenylate Like 2 (Oasl2) knock out mouse.
Authors: Ghosh Arundhati , Frank David Anthony , Mosch Nora , Sarkar Saumendra N
Published: January 2023

Our immune system works constantly to keep us safe from invading viruses and bacteria. It can even prevent us from developing cancer. Among these immune cells, macrophages can recognize these foreign threats and neutralize them. Macrophages can be differentiated into specific phenotypes such as M1 and M2 with specific signals. M1 and M2 macrophages each secrete specific cytokines and chemokines promote proinflammatory and antiinflammatory responses in host cells respectively. 2′-5′ oligoadenylate like protein (OASL) protein belongs to a well known family of antiviral proteins knows as Oligoadenylate Synthetase. Both human OASL and mouse isoform Oasl2 perform similar functions in inhibiting RNA virus multiplication within host cells which has been characterized in detail. In the present work, we have characterized the properties of Bone marrow derived macrophages (BMDM) of Wild type (Wt) and Oasl2 knock out (KO) mice in terms of their polarization to M1 and M2 phenotypes. Detailed analysis and comparison of polarized M1 and M2 macrophages from Wt and KO mouse revealed different behavior of KO mouse M1 and M2 macrophages compared to Wt. KO M1 macrophages have more induction of IRF1 resulting more production and secretion of IL-1β in to the culture media. On the other hand, KO M2 produced more Arginase I within the cells. Detail studies of properties of macrophages from Oasl2 KO mice will help us understand the function of this protein within body.
In vivo validation of a novel anti-mesothelin nanobody for early detection of mesothelin-expressing cancers
Authors: Scholler Nathalie , Yin Catherine , sharma khushboo , Prantner Andrew M , Kamat kalika , Repellin Claire , Sambucetti Lidia
Published: January 2023

Antibodies and antibody fragments are widely used in oncology for nanotechnology-based diagnostic, therapeutic, and prognostic assays. Recombinant antibodies that recognize mesothelin, a differentially expressed cancer biomarker with limited normal expression that is upregulated in a variety of epithelial tumors, are important for developing next generation diagnostic or therapeutic platforms and immunosensors because of the flexibility to incorporate various tags or functional groups for site-specific and oriented attachment of antibody fragments to surfaces, labeling reagents or therapeutics. We exploited the inherent stability of camelid heavy chain to develop a site-specific biotinylated, flag-tagged nanobody that recognizes human and mouse mesothelin. We previously published the in vitro characterization of the anti-mesothelin nanobody G3a and then we sought to address whether G3a nanobody could detect mesothelin-expressing tumors in syngeneic and xenograft mouse models of ovarian or lung cancers. Tumor cells were injected subcutaneously (A1847), IV (EKVX, H460, A549), IP or intra ovary (Luc-ID8), and detected at different time points by in vivo imaging (IVIS) or by magnetic resonance imaging (MRI). For in vivo imaging, G3a nanobody was coupled to fluorescently-labelled streptavidin (IRB680W); for MRI, G3a was coupled to avidin-coated magnetic beads. We found that G3a nanobody generated robust and highly mesothelin-specific signals that permitted the detection of very low tumor burden barely detectable by luciferin. Our results indicate that G3a is a valuable reagent for early detection of mesothelin-expressing cancers, as well as a promising delivery agent for imaging and therapeutic conjugates.
Expression of adipocyte/macrophage fatty acid binding protein in tumor associated macrophage promotes breast/mammary tumor progression
Authors: Hao Jiaqing , Yan Fei , Zhang Yuwen , Triplett Ashley , Zhang Ying , Schultz Debra A. , Sun Yanwen , Zeng Jun , Silverstein Kevin A.T. , Zheng Qi , Bernlohr David , Cleary Margot P. , Egilmez Nejat K. , Sauter Edward , Liu Shujun , Suttles Jill , Li Bing
Published: January 2023

Tumor associated macrophages (TAMs) play a critical role in cancer development and progression. However, due to the heterogeneity of TAMs, it remains a major challenge to identify clinically-relevant markers for pro-tumor TAMs. Here, we report that expression of adipocyte/macrophage fatty acid binding protein (A-FABP) in TAMs promotes breast cancer progression. While upregulation of A-FABP was inversely associated with breast cancer survival, deficiency of A-FABP significantly reduced mammary tumor growth and metastasis. Furthermore, we demonstrated that the pro-tumor effect of A-FABP was mediated by TAMs. More specifically, A-FABP was preferentially upregulated in a subset of TAMs that exhibited a CD11b+F4/80+MHCII−Ly6C− phenotype, which promoted tumor progression through IL-6/STAT3 pathway. Mechanistically, activated NFκB in A-FABP+ TAMs repressed the expression of miR-29b, resulting in enhanced tumor-promoting IL-6 signaling as compared to A-FABP −/− TAMs. Thus, A-FABP represents as a new functional marker for pro-tumor TAMs and a new target for macrophage-based tumor immunotherapy.
Mechanisms of PTEN inactivation and pAKT dependence in ATM-deficient thymic T cell lymphomas
Authors: An Joseph Byung Hoon , Hathcock Karen S , Hodes Richard J
Published: January 2023

Ataxia telangiectasia mutated (ATM) is a serine-threonine kinase that is essential for maintaining genomic integrity in response to DNA double-strand breaks. AT patients and ATM-deficient mice develop lymphoid malignancies at high frequency, establishing ATM’s role as a tumor suppressor. It was previously reported that murine Atm−/− T cell lymphomas have a recurrent loss of the gene encoding phosphatase and tensin homolog (PTEN), a tumor suppressor that plays a critical role in inhibiting the PI3K/AKT/mTOR pathway. Our analyses of Atm−/− T cell lymphomas revealed that 12/13 express high levels of phospho-AKT (pAKT). This upregulation of AKT signaling is consistent with the finding that 11/12 pAKThigh tumors lack PTEN protein. One tumor was PTEN+ but failed to downregulate pAKT. Here, we study the Atm−/− tumor model in order to uncover the mechanism by which PTEN expression is ablated. Interestingly, our analyses found that 9/11 PTEN-deficient cell lines have incurred a variety of alterations including exonal deletions of pten or truncations of its mRNA, providing a basis for PTEN loss-of-function. The remaining three lines, however, possess full-length Pten exomes and transcripts. Sequencing the Pten mRNA of these cell lines is in progress to identify mechanisms responsible for the absence of PTEN protein. We have further observed that in vitro treatment of Atm−/− T cell lymphomas with the pAKT inhibitor MK2206 resulted in substantial impairment of survival, consistent with their dependence on activated AKT in the absence of regulation by PTEN. Taken together, these results demonstrate that the majority of Atm−/− T cell lymphomas lose PTEN function through multiple mechanisms and become dependent on AKT signaling for their survival.
A sand fly salivary chemotactic protein drives neutrophil migration and exacerbates Leishmania infection
Authors: Guimaraes Anderson , Wen Xi , Castro Waldione , Townsend Shannon , Brzostowski Joseph , Meneses Claudio , Kamhawi Shaden , Valenzuela Jesus , Oliveira Fabiano
Published: January 2023

Neutrophils are the first cells recruited to the site of an infected sand fly bite and interact with Leishmania parasites. Here we investigate the effect of sand fly salivary proteins on neutrophil recruitment. We purified bone marrow neutrophils from C57BL/6 mice or from peripheral blood of healthy human or dog donors. Chemotaxis driven by sand fly saliva of vectors of visceral (Lutzomyia longipalpis) or cutaneous (Phlebotomus duboscqi) disease was measured by modified Boyden chamber assay and by imaging with the EZTAXIScan assay. Neutrophils migrated towards saliva glands of the vectors P. duboscqi and L. longipalpis, in a dose-dependent manner. Proteinase K treatment of saliva completely abrogated neutrophil recruitment, suggesting a protein as the chemotactic factor. To unearth the identity of the chemotactic protein, we generated plasmids coding for the 20 most abundant salivary proteins from P. duboscqi and injected them in the ears of C57BL/6 mice. Neutrophil recruitment was analyzed by flow cytometry staining. We identified 2 different salivary protein-coding plasmids with neutrophil recruitment activity. We produced these 2 recombinant proteins in the HEK293 cell line system and we observed that a family of 40kDa salivary proteins acts as chemokines for neutrophils both in our in vitro and in vivo assays. Moreover, the coinjection of Leishmania major parasites with the recombinant proteins in the ear of C57BL/6 mice exacerbated the infection. We plan to test if neutralization of the salivary chemotactic protein can alter Leishmania infection. We put forward the hypothesis that blockage of this protein will disrupt neutrophil migration early in the Leishmania infection possibly altering the disease outcome.
Metabolic reprogramming of CD4 T cells exacerbates Herpes Stromal Keratitis
Authors: Varanasi Siva Karthik , Rouse Barry T
Published: January 2023

Ocular infection with Herpes Simplex Virus type 1 (HSV-1) sets off an inflammatory reaction in the cornea which leads to both virus clearance as well as chronic lesions that are orchestrated by CD4 T cells. Effector CD4+ T cells rely on glycolysis for inflammatory effector functions and Treg rely on mitochondrial metabolism. How these processes contribute to Herpes stromal keratitis (HSK) is unclear. We show that glycolysis is elevated in CD4+ T cells from mice with HSK as compared to non-immunized controls. In addition, glucose uptake by CD4 T cells at the lesion site and draining lymph nodes were higher in animals with HSK. In vitro, T cells activation led to enhanced glycolysis, which was inhibited by inhibitor 2-deoxy-D-glucose (2DG). Th1 generated in vitro displayed enhanced glycolysis rates compared to Treg cells. Hence, inhibition of glycolysis by 2DG inhibited the differentiation and function of interferon-γ (IFN-γ) producing Th1 cells, largely un-affecting Treg cells. In vivo, treatment of animals with 2DG normalized T cell metabolism rebalancing the Treg to Th1 ratio and limited the SK lesion progression. Furthermore, animals infected with HSV-1 displayed enhanced blood glucose levels at time points when the inflammation at draining lymph nodes and lesion site are at its peak, which may both be the cause and effect of the inflammation. These results suggest that normalization of T cell metabolism through inhibition of glycolysis is a promising therapeutic venue for HSK.
Impaired CD8 T cell response to viral infection following acute spinal cord injury
Authors: Norden Diana Maria , Bethea John R , Jiang Jiu
Published: January 2023

Trauma to the spinal cord disrupts essential communication between the central nervous system and the immune system. This is highly important as patients with spinal cord injury (SCI) are at greater risk for infections that increase morbidity and mortality. Previous studies have confirmed immune depression in mouse models of chronic SCI. In mice, injury at the high thoracic level disrupts sympathetic output and leads to sustained elevations of norepinephrine in the spleen. SCI patients, however, are at high risk for infections acutely after SCI while hospitalized. Here, we induced a mid thoracic injury where integrity of the sympathetic response is maintained and investigated the antiviral response to influenza an acute timepoint. Mice received a 70kdyn contusion injury at T9 and were challenged intranasally with influenza virus (HKx31, H3N2) 7 days after injury. Following infection, injured mice had a diminished CD8 T cell response with decreased numbers of virus-specific CD8 cells in the lungs. Effector functions were also impaired in injured mice. Expansion of CD62Llow/CD44high effector CD8 T cells was reduced in injured mice and recruitment of CD8 T cells to the lungs was decreased compared to uninjured mice. In addition, the number of intracellular IFNγ-secreting CD8 T cells after in vitro stimulation with virus-specific peptide was reduced in both spleen and lung of injured mice. Overall, these data show an impaired CD8 T cell response to viral infection after acute SCI and suggest that immune depression following SCI occurs rapidly and may be independent of chronic norepinephrine elevations.
Enhancement of antigen presentation of a vaccine with the use of human anti-rhamnose antibody both in-vitro and in-vivo
Authors: Hossain Md Kamal , Vartak Abhishek , Karmakar Partha , Sucheck Steven J. , Wall Katherine Ann
Published: January 2023

Since anti rhamnose antibody is the most common natural anti-carbohydrate antibody present in human serum, using rhamnose as a targeting moiety for a rhamnose containing vaccine can be a good approach for effective vaccine formulation. Binding rhamnose of a rhamnose-containing vaccine with anti-rhamnose antibody has been proven effective for capturing and processing of antigen by antigen presenting cells (APC) via stimulatory Fc receptors. This was shown using anti-rhamnose antibodies generated in mice. We wished to determine if human anti-rhamnose antibodies isolated from human serum work in a similar manner. To isolate anti-rhamnose antibodies, we prepared a rhamnose affinity column with a rhamonse-2 amino ethyl linker conjugate coupled to CNBr-activated Sepharose. In-vitro, human anti-rhamnose antibodies enhanced uptake of rhamnose-ovalbumin by murine APC as determined by proliferation of ovalbumin-primed T cells. In-vivo, injection of human anti-rhamnose antibodies into mice improved the priming of T cells to rhamnose-ovalbumin. Also, we found increased priming of both CD4+ and CD8+ T cells toward the MUC1-Tn cancer antigen when mice were given human anti-rhamnose antibodies prior to a rhamnose-modified MUC1-Tn-containing cancer vaccine. Other human immunoglobulins did not enhance priming. Therefore, we have confirmed that human anti-rhamnose antibodies can enhance immune responses to rhamnose-containing vaccines through increased antigen uptake and presentation.
New strategies in vaccine design for the induction of CD8 T cell responses using biodegradable iron oxide nanoparticles
Authors: Powles Liam M , Wilson Kirsty , Xiang Sue D , Selomulya Cordelia , Plebanski Magdalena
Published: January 2023

For vaccines targeting diseases such as malaria and cancer, CD8 T cell responses are vital. Traditional adjuvants, like alum, are incapable of inducing these responses and thus new delivery strategies are required. Previously, we have shown that biocompatible polystyrene nanoparticles (PSNPs) (carboxylate, 40–50 nm) can induce potent cytotoxic T cell and antibody responses when conjugated to antigen and can protect mice against experimental tumours or clear large established tumours after a single immunization. As a biodegradable alternative; we synthesised ~50 nm iron oxide nanoparticles (IONPs) stabilized with the polysaccharide pullulan. The IONPs share similar characteristics (size, charge and shape) with the PSNPs, are stable, non-toxic, rapidly taken up by dendritic cells in vitro and are considered inert as they do not upregulate costimulatory markers.

The adjuvant/carrier capabilities of IONPs when conjugated to model antigen ovalbumin (OVA) were assessed in vivo. IONPs can induce strong antibody responses but failed to boost the T cell response (Th1 and Th2) after two immunisations, despite their similarity to PSNPs. This implies that the composition of a NP can affect its interactions with the immune system and as such its behaviour as a carrier. To produce an IONP delivery system in which CD8 T cells could also be induced in addition to strong antibody responses, IONPs were co-delivered with the TLR9 agonist CpG. This led to > 2-fold increases in CD8 T cell and antibody responses above either IONPs or CpG alone. Therefore, these biodegradable particles have the capacity to synergize with an inflammatory adjuvant and provide a new strategy for vaccine delivery focused on the induction of CD8 T cell responses.
Induction of stem-like memory T cells by cancer vaccine is associated with TCR downregulation and correlates to strong antitumor effect
Authors: He Yukai , Wu Sha , Zhu Wei , Peng Yibing , Wang Lan , Hong Yuan , Fu Ning
Published: January 2023

To understand why vaccine-activated tumor-specific T cells often fail to generate antitumor effects, we have focused on studying the intrinsic attributes of two alpha fetoprotein-specific CD8 T cells (Tet212 and Tet499). We found that while Tet499 survived in vitro re-stimulation and maintained their functions, Tet212 underwent exhaustion and apoptosis. In vivo, Tet499 generated more expansion than Tet212 and stronger antitumor effects. The different antigen responsiveness and antitumor effects of Tet212 and Tet499 correlated to their activation and differentiation state. Compared to Tet212, Tet499 were in a lower activation state and contained more stem-like memory T cells (Tscm) that could undergo more expansion in vivo. Mechanistically, Tet499 TCR was more downregulated than Tet212 TCR, which may be responsible for the lower activation and differentiation state of Tet499. In contrast to the notion that TCR downregulation dampens T cell antigen reactivity, our data show that it may help T cells maintain their antigen reactivity by halting their activation and differentiation at Tscm stage and by protecting T effector cells from antigen-induced exhaustion and apoptosis. These findings indicate that the focus of cancer vaccination should be on eliciting tumor-specific T cells at earlier differentiation stage including Tscm for better antigen reactivity.
Immune checkpoint function of T cell-derived CD70 in inflammatory T cell responses
Authors: ONeill Rachel , Du Wei , Alquassim Emad , Mohammadpour Hemn , Qiu Jingxin , Chen George , McCarthy Philip , Cao Xuefang
Published: January 2023

The CD27-CD70 pathway is known to provide a costimulatory signal with CD70 expressed on antigen-presenting cells while CD27 functioning on T cells. Although CD70 is also expressed on activated T cells, it remains unclear how T cell-derived CD70 affects T cell function. Therefore, we have assessed the role of T cell intrinsic CD70 using multiple models including autoimmune inflammatory bowel disease (IBD) and allogeneic graft-versus-host disease (GVHD). Compared with WT T cells, CD70−/− T cells surprisingly caused more severe IBD and GVHD and produced higher levels of inflammatory cytokines. Mechanistic analyses reveal that IFNg induces CD70 expression in T cells, and CD70 limits T cell expansion via a regulatory T cell-independent mechanism that involves caspase-dependent T cell apoptosis and upregulation of other immune checkpoint molecules. Overall, our findings demonstrate that T cell-derived CD70 plays a novel immune checkpoint role in inhibiting inflammatory T cell responses.
Vio®Dyes meet REAfinity - High Resolution Antibody-Fluorochrome Conjugates for Multi-Parameter Flow Cytometry Analysis
Authors: Evaristo Cesar , Vahldieck Marco , Krauthaeuser Susanne , Jansen Nicole , Richter Anne , Dose Christian
Published: January 2023

State-of-the-art sensitivity and target specificity of fluorochrome-conjugated antibodies limit applications in flow cytometry. Identification of cell subsets can be challenging due to low expression levels of surface and intracellular markers. The combination of Vio®Dyes and REAfinity™ antibodies provides researchers with the most powerful and reliable tools for flow cytometric cells analysis.

REAfinity™ are recombinant antibodies of human IgG1 isotype with a mutated region within the Fc part, which prevents Fcγ receptor interactions. They are characterized by high antigen specificity and consistent quality compared to conventional hybridoma-derived monoclonal antibodies. Vio®Dyes, e.g. Vio®515, VioBright®515, Vio®667, VioBright®667, and PE-Vio®615, allow due to their excellent fluorescent properties the detection of low expressed markers and provide new choices for multi-parameter analysis. To demonstrate the potential of the combination of REAfinity™ antibodies and Vio®Dyes, we developed detection panels and protocols for the characterization of FOXP3+, HELIOS+, and CD45RA+ in CD4+ CD25+ CD127low/− human regulatory T cells (Tregs). Tregs were detected and enumerated in whole blood, PBMC, and in samples of magnetically enriched Tregs utilizing the newly developed reagents. In addition, we set-up new and fast staining protocols using 96-well plates or magnetic columns, which allow improved identification of Treg subsets.

In summary, the portfolio of Vio®Dyes conjugated REAfinity and special protocols open up new ways for designing and implementing multicolor panels towards highly sensitive, reproducible, and rapid cell profiling approaches.
Human Th17 activity profile in oral chronic Graft-versus-Host Disease
Authors: Dodge Joshua , da Silva Ana C Costa , Dhamala Susan , Rose Jeremy J , Pavletic Steven Z , Hakim Frances T , Mays Jacqueline W.
Published: January 2023

Immunotherapy using allogeneic hematopoietic stem cell transplantation (alloHSCT) is highly effective; however, its use is limited by the incidence of chronic graft-versus-host disease (cGVHD). The oral cavity, which is affected by cGVHD, is a highly accessible mucosal site with its own local biofluid – saliva – but little is known about the pathogenic progression of cGVHD in oral tissues. This study examined the relationship between salivary gland gene expression, salivary proteins and the immune cell network within oral tissues at onset of oral cGVHD.

Patients enrolled in NIH cGVHD trials (NCT00331968, NCT00520130, NCT01851382) were evaluated after cGVHD onset. Whole saliva from individual oral cGVHD patients (n=58) and healthy controls (n=10) was analyzed for a 13-plex panel of immune-related analytes and revealed elevated expression of tissue remodeling factors and IFN- and IL-17-induced chemokines that correlated with clinical scoring of cGVHD severity in a multivariate analysis. Microarray of patient MSG indicated canonical pathway expression including innate immune pathways, and IFN, IL-17 and IL-22-induced processes. Confocal microscopy confirmed the presence of immune cells in oral tissues. Extending our laboratory’s prior observations of Th1 effectors in BM, Th17 cells (CD4+/CD161+/IL-17+) and Type I IFN-induced factors were identified in the majority of available BM and MSG of cGVHD patients, but not in unaffected post-transplant controls.

Current analyses in cGVHD patient samples implicate activation of IFN pathways and presence of Th17 cells in oral tissues in the pathogenesis of oral cGVHD, though the specific sequence and role of these cells in salivary gland pathogenesis during cGVHD remain to be determined.
Human natural killer cell expansion in vitro using mitomycin-C treatment as an alternative to irradiation of modified K562 feeder cells.
Authors: Carr William H , Zinsou Carmen , Rowe Tahishia , Lange Christopher , Lawal Oluwadamilola O.
Published: January 2023

Introduction

Current methods for in vitro expansion of Natural Killer (NK) cells require feeder cell irradiation, which is not readily available in many biomedical research or clinical facilities. Here we evaluated mitomycin-C (MMC) treatment of feeder cells as an alternative to irradiation for the purpose of expanding NK cells in human peripheral blood mononuclear cell (PBMC) cultures.

Methods

Using PBMCs from 4 healthy blood donors and genetically modified K562 feeder cells (K562-mb21-41BBL cells) that preferentially stimulate NK cells, we evaluated the efficacy of MMC treatment in limiting the proliferation of the feeder cells at a range of doses (0 ug/mL, 2 ug/mL, 10 ug/mL, 20 ug/mL, 30 ug/mL, and 200 ug/mL) with 3-hour incubation. We used 7AAD dye and CFSE proliferation dye to measure viability and cell proliferation, respectively. We then compared the percentages of NK cells (CD3neg, CD56pos) in PBMCs cultured for 14 days in the presence of MMC-treated feeder cells or irradiated feeder cells using multiparametric flow cytometry.

Results

We found that 10 ug/mL dose of MMC for 3-hrs achieved a significantly higher percentage of viable cells compared to higher doses (p<0.05, ANOVA), and also significantly limited proliferation compared to untreated feeder cells. Furthermore we found no significant difference in NK cell expansion by 10 ug/mL MMC treated feeder cells and 100 grays irradiated feeder cells.

Discussion

In conclusion, we found that 10 ug/mL mitomycin-C treatment of modified K562 feeder cells was optimal for limiting their proliferation for NK cell expansion that was equivalent to using irradiated feeder cells.
Identification of kidney CD45intCD11bintF4/80+MHCII+Ly6C− macrophages initially masquerading as T cells
Authors: Lee Sul A , Noel Sanjeev , Sadasivam Mohanraj , Hamad Abdel Rahim , Rabb Hamid
Published: January 2023

Background

Mononuclear phagocytic cells (MPCs) have important roles in the pathogenesis of many kidney diseases, including ischemia-reperfusion injury (IRI) and allograft rejection. Despite the recent advances on description of MPC subpopulations and their functional characterization in kidney, there is no clear consensus for the classification of MPC subpopulations. Here we describe a newly identified renal macrophage, named CD45intCD11bint cells.

Methods

Kidney mononuclear cells were isolated from C57BL6 male mice under steady state and after IRI and analyzed using flow cytometry.

Results

While focusing on TCRαβ+CD4−CD8− kidney T cells, we identified a cell population that binds only to TCRβ and CD8β antibodies but not to CD8α antibody. Further studies using Fc receptor blockers disclosed that this population was a renal macrophage subset which was different from other macrophages by its intermediate expression of CD45 and CD11b. These CD45intCD11bint macrophages were further characterized as F4/80+MHCII+Ly6C− cells comprising 40% of MPCs in normal kidney. CD45intCD11bint macrophages are found predominantly in the kidney as compared to other lymphoid and non-lymphoid organs (ANOVA, p = 0.002). In addition, CD45intCD11bint population significantly decreased 48 hours after IRI in contrast to steady increase of CD45high macrophages (p = 0.027). Relevance of this population to human kidney is being investigated.

Conclusion

Kidney CD45intCD11bintF4/80+MHCII+Ly6C− macrophages are newly defined MPCs that comprise a major subset of resident renal MPCs. They have unique surface phenotype as well as T-cell mimicking characteristics. This new subset could play an important role in immune diseases of the kidney.
Regulatory B cells in the blood of plasma cell hepatitis patients who have undergone liver transplantation
Authors: Hasan Md Mahmudul , Joo HyeMee , Klintmalm Goran B. , Oh SangKon , Thompson-Snipes LuAnn
Published: January 2023

Alloreactive T cells and antibody-mediated rejection (AMR) appear to be barriers to long-term organ engraftment. Regulatory B cells (Bregs), a B cell subset, can protect against autoimmune/inflammatory damage, in part, by producing IL-10, an anti-inflammatory cytokine. However, the role of Bregs in solid organ transplantation is poorly understood. In this study, we examined IL-10 producing human Bregs in the blood of healthy controls compared to liver transplanted patients with plasma cell hepatitis (PCH), a variant of late onset rejection. First, we optimized conditions for human Breg differentiation in vitro. Based on the presence of differentially expressed cell surface markers on IL-10+ CD19+ B cells, we then established a profile of Bregs from several human tissues including tonsils and spleens and demonstrated that in vitro generated IL-10+CD19+ B cells are capable to suppress alloantigen stimulated T cell proliferation and cytokine production. Finally, we find that the frequency of CpG B activated IL-10+CD19+ B cells is negligible in PCH+ liver transplant patients compared with healthy controls. In line with this, the frequency of transitional B cells (CD19+CD24hiCD38hi) is significantly lower in PCH+ liver transplanted patients than healthy controls. Our findings suggest that a lack of immunosuppressive Bregs could be associated with the rejection in the PCH+ patients
The Intracutaneous Absorption of Antigen
Authors: Smith G. H. , Cook M. W.
Published: January 2023

That the body is capable of reacting to parenterally introduced antigenic substances has been amply proven, but as yet there has been no satisfactory explanation of the mechanism of the interaction, that is, it has not been conclusively determined whether antigen is eliminated as antigen or whether it enters into the metabolic processes and becomes utilized or denatured. It can not be assumed that identical processes follow the introduction of different antigens or even of the same antigen when injected by different routes. In fact, opinion differs very widely as to what happens when a given antigen is introduced by a single route. Further complications arise through the fact that animals receiving antigen may, as a result of inherent peculiarities or experimental procedure, be endowed with a variety of metabolic forces, which in individual cases may so combine as to give a variety of results.
Experiments with a Possible Bearing Upon the So-Called Non-Specific, Intravenous Vaccine Therapy
Authors: Teague Oscar , McWilliams Helen I.
Published: January 2023

The experimental foundation for a non-specific vaccine therapy was laid by Emmerich (1) thirty years ago. He showed that rabbits inoculated with streptococci possessed a certain amount of immunity against anthrax. He also observed that an anthrax infection in rabbits, even after it had called forth distinct symptoms, could be favorably influenced by the injection of streptococci—particularly if the latter injection was administered intravenously. Pawlovsky (2) confirmed these results, and demonstrated that Friedländer's bacillus and B. prodigiosus also retarded the development of the anthrax infection. Bouchard (3) found that B. pyocyaneus could be used for this purpose. Then Freudenreich (4) and Woodhead and Cartwright Wood (5) obtained similar results with killed cultures of B. pyocyaneus, and Buchner (6), with killed cultures of Friedländer's bacillus. Von Dungern (7) injected killed cultures of Friedländer's bacillus intravenously and observed that rabbits so treated were more resistant to anthrax bacilli inoculated subcutaneously one or several days later, than were normal rabbits.
The Differentiation of a Type of Human Blood by Means of Normal Animal Serum
Authors: Landsteiner K. , Levine Philip
Published: January 2023

The main result of the present investigation consists in making available a method—namely, the use of normal animal serum—for readily differentiating a certain rather frequent type of human blood designated as P, whereas the reagents previously employed for this purpose were not easily accessible.

The method may be of use for studies on heredity and racial distribution.
The Antigenic Properties of Fibrinogens
Authors: Kyes Preston , Porter Robert T.
Published: January 2023

Previous publications (1) (2) from this laboratory have pointed out the inadequacy of the usual methods of blood defibrination in preparing fibrinogen-free serum and particular methods have been given by which such a serum may be obtained. The fact has also been established that traces of fibrinogen in a serum profoundly modify the immune reactions of that serum both in vivo and in vitro, provoking shock reactions in the former and pseudoprecipitin and agglutinin reactions in the latter.

Continuing the study of fibrinogen in relation to immune reactions we have recently tested the antigenic properties of fibrinogen and this paper presents evidence that the injection of foreign fibrinogen into an animal induces the production of antibodies specific for the particular fibrinogen used.

The fibrinogen chiefly employed in our experiments was prepared from well developed Plymouth Rock cockerels.
Comparative Study of the Anaphylactic and Tuberculin Types of Hypersensitiveness
Authors: Dienes L.
Published: January 2023

The crystalline egg albumin and egg globulin differ considerably in their relationship to the different manifestations of hypersensitiveness. The crystalline egg albumin tends to sensitize the organism to those manifestations of the hypersensitiveness which are mediated by antibodies. The egg globulin tends to sensitize to those in which the active immunization plays the more important rôle. The difference between the antigens lies in their capacity to produce a different type of sensitization. We find no marked difference between them concerning their capacity to produce antibodies or to elicit the reactions themselves. The observations with crystalline egg albumin and egg globulins present an analogy to the behavior of certain bacterial antigens which we usually find connected either with the tuberculin or with the anaphylactic type of sensitiveness.

We have given a short review of our experiences concerning the capacity of different antigens to produce the tuberculin type of skin sensitiveness. With all innocuous antigens which we tested—as eggwhite and animal sera—we succeeded in producing this sensitiveness. With bacterial antigens we have not so far succeeded in producing it.