bluuebunny/crossref_metadata_2025 huggingface dataset:
Macrophage heterogeneity in human macrophages correlates with autophagy-dependent anti-mycobacterial activity and ability to present antigen to CD4 T cells
Authors: Khan Arshad , Mann Lovepreet , Papanna Ramesha , Won Jong Hak , Mani Arunmani , Canaday David , Jagannath Chinnaswamy
Published: January 2023

Tuberculosis due to Mycobacterium tuberculosis (Mtb) kills around 1.5 million people each year. Although only 10% of humans develop active disease after aerosol infection, the basis for differential susceptibility of humans remains unclear. To determine if human macrophages are heterogeneous in their ability to kill Mtb, cord blood derived macrophages were treated with either IFN-γ or IL-4 to prepare M1 and M2 phenotype in vitro. Human THP-1 macrophages were also similarly prepared. Macrophages were infected with a 1:1 ratio of Mtb, incubated and on days 3 and 5 post infection, macrophage lysates were plated for CFU counts. In addition, intracellular autophagy was measured in gfpMtb infected macrophages using cytoID stains to detect autophagosomes and LC3 lipidation through western blot of macrophage lysates. Finally, Mtb infected macrophages were overlaid using CD4 T cells specific for the Antigen-85B epitope to measure antigen presentation. M1 macrophages showed a bacteriostatic ability and restricted the growth of Mtb while IL-4 treated M2 macrophages showed nearly 1 log increase in the growth of Mtb. Thus M2 macrophages were permissive for growth of Mtb. Consistent with this observation, gfpMtb co-localized with autophagosomes more frequently in M1 macrophages suggesting a role for autophagy during growth inhibition. LC3 lipidation was better in M1 compared to M2 macrophages. Finally, M1 macrophages presented Ag85B to CD4 T cells better than M2 macrophages. These observations suggest that macrophage heterogeneity into M1 and M2 is associated with an innate ability to kill intracellular tuberculosis and trigger immune activation.
TLT-1 as a possible modulator of Systemic Lupus Erythematosus.
Authors: Morales Jessica , Rodriguez Yerania , Vila Karina , Ricaurte Martha , Vila Luis M , Washington Anthony V.
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by immune system hyperactivity and autoantibody production that can affect almost any organ system. TREM like transcript-1 (TLT-1) is a membrane receptor found in platelet α-granules with a soluble form (sTLT-1) detected in serum but not plasma of healthy individuals. The evaluations of patients suffering from diseases, such as sepsis, show a significant increase of plasma sTLT-1 levels. It is not known if TLT-1 plays a role in auto immune diseases such as Lupus. In the present study we evaluate the potential that TLT-1 may play a role in the etiology of SLE. We assessed the plasma of 46 SLE patients and compared to 28 healthy controls by ELISA. Our results showed that SLE patients had a significant decrease in plasma sTLT-1 levels compared to healthy controls (9.0 [7.2] vs. 18.6 [22.3] ng/ml, p=0.008). A negative correlation was observed between TLT-1 levels and LupusPRO [lupus symptoms (r = −0.388, p = 0.055). Based on this data; we hypothesized that SLE patients produce autoantibodies against TLT-1 in serum which might correlate with clinical aspects of the disease. To address this question; TLT-1 antibodies levels in serum were measured by ELISA. Our data suggests that a sub-population of SLE patients have TLT-1 autoantibodies. This data was further confirmed by confocal microscopy. Using HEK cells stably transfected with TLT-1; we evaluated the serum of SLE patients for the presence of TLT-1 autoantibodies using immunofluorescence. Our data confirms that a sub-population of SLE patients with active disease expresses antibodies against TLT-1 suggesting a possible role for TLT-1 during the pathogenesis of SLE and targets new treatment possibilities for lupus patients.
Cellular gatekeepers of the humoral immune response
Authors: Yousif Ashraf S , Omatsu Tatsushi , Weaver Grant , Shah Pankaj , Reinnecker Hans-Christian , Lingwood Daniel
Published: January 2023

The spleen is critical for eliminating bloodborne pathogens and preventing systemic infection. In the marginal zone (MZ), the site in which splenic antibody responses are first initiated, we have discovered a dendritic cell (DC) ‘hairnet’ architecture that is responsible for extracting two unrelated viral antigens from the perfusing blood and importing them into B cell follicle to initiate IgM and IgG responses. The IgM response was independent of T-cell help, and proceeded via DC display of conformationally intact antigen in the absence of MHC proteins. Interestingly the DC-antigen captures and importing mechanism are solely governed by the glycan of the antigens. Taking all together, we propose that DC hairnet architecture, inshealthing the MZ, constitutes a filtering device that capture and concentrates protein antigens in a glycan-dependent manner from solution and catalyzes their delivery to B cells follicle to initiate humoral immune responses.
Human myeloid derived suppressor cell (MDSC) subset phenotypes
Authors: Talmadge James E , Cole Kathryn , Britton Holly , Dafferner Alicia , Warkentin Phyllis
Published: January 2023

MDSCs are increased in cancer patients and were originally defined based on T-cell suppression and a negative correlation with prognosis and overall survival and a direct correlation with tumor stage. They were originally described, based on CD34 expression and a hematopoietic progenitor phenotype. However, their phenotypic definition is investigator dependent in association with suppressor cell function. There are two main subsets defined based on a lack of lymphoid and differential expression of myeloid markers resulting in a cellular continuum from multi-potential hematopoietic progenitors to differentiated granulocytes and monocytes. One recent hypothesis is that MDSCs form three subsets including macrophage MDSC (CD14+ and no CD15 or CD66b); granulocytic MDSCs (CD15 and/or CD66b expression and no CD14); and immature (i-) MDSC (linage (Lin) negative, CD33+ cells). Unfortunately, there is no consensus on markers extent to CD14 and CD15 with linage and HLA-DR negativity providing a frequent; although not universal theme. We report a flow cytometry gating strategy utilizing a single staining tube and a linear phenotypic characterization of MDSC subsets supported by density and T-cell suppression. We suggest that staining with antibodies to Lin, HLA-DR, CD14, CD15, CD11b and CD33 is sufficient to subset into the three phenotypes that can be further subset based on CD16. PDL1 and CD34 expression. The majority of iMDSC are CD34+CD33+ cells supporting a plastic phenotype and myeloid commitment and can be further, delineated based on CD16 expression supporting immaturity. We note, that some cancer patients express MDSCs at high levels; although the subsets preponderance varies between patients.
Scavenger receptor CD36 on B cells senses modified self-antigens to prevent autoimmunity
Authors: Duhlin Amanda , Grasset Emilie K , He Chenfei , Amara Khaled , Sippl Natalie , Lindh Emma , Vargas Leonardo , Dahlberg Carin , Westerberg Lisa , Smith Edvard CI , Malmström Vivianne , Pierce Susan K , Karlsson Mikael C
Published: January 2023

Scavenger receptor CD36 has been shown to mediate uptake of modified self-antigens such as apoptotic cells and oxidized LDL in macrophages and dendritic cells. Interestingly, CD36 was discovered to be preferentially expressed also on an innate B cell subtype, marginal zone B cells (MZB). Here we investigate the role of CD36 in B cell activation in the context of apoptotic cell clearance and break of tolerance to self. We found that formation of germinal center B cells and autoantibody production in response to an increased load of apoptotic cells coincide with downregulation of CD36 on MZB. We could also in connection to this show that systemic lupus erythematosus (SLE) patients have lower levels of peripheral MZB compared to healthy individuals. B cells lacking CD36 proved more easily activated in response to apoptotic cells, as was shown in bone marrow (BM) chimeras reconstituted with wild type and CD36-deficient BM. In steady-state, we found that CD36 co-localizes with the B cell receptor and the tyrosine kinase Lyn. Interestingly, upon cross-linking of the inhibitory Fc receptor, FcgRIIb, CD36 instead co-localizes with this receptor. This suggests that CD36 interacts with FcgRIIb to activate a negative signaling cascade in B cells. Our data implicate CD36 in the early regulation of B cell responses towards apoptotic cells and autoantibody production. In line with this we find that CD36-deficient B cells are more resistant to FcgRIIb-induced cell death. Thus, CD36 exerts its regulatory function by associating with a known negative signaling pathway in B cells involving Lyn and FcgRIIb. This finding could have implications for autoimmune diseases involving apoptotic cell-derived self-antigens, such as SLE.
IL-12 stimulates CTLs to secrete exosomes with biological functions
Authors: Xiao Zhengguo , Wang Yan , Li Lei
Published: January 2023

An effective CTL response is generally accomplished by massive CTL expansion and production of effector molecules, potentially exhausting resources and rendering the host vulnerable to other infections. It is not yet well known how the host maintains relative functional immunity against other pathogens during the course of an ongoing, robust CTL response against an active infection. We found that CTLs secrete exosomes in the presence and absence of IL-12, which all contain typical exosomal markers, such as ALIX and TSG101, as well as CTL proteins ZAP70 and granzyme B (GZB). However, when conditioned with IL-12, CTLs release smaller exosomes, which contain more proteins of exosomal markers, compared with exosomes secreted by control CTLs. More importantly, IL-12-conditioned exosomes can directly stimulate naïve CTLs to produce IFNγ, TNFα and GZB, which accompanied by upregulation of CD25, CD69 and CD44, independent of antigen or cytokines, whereas exosomes from control CTLs have minimal effects. In addition, IL-12-conditioned exosomes are able to strengthen weak TCR stimulation in CTLs. Proteomic analysis demonstrates that IL-12 stimulation skews exosomal proteins toward certain metabolism-related enzyme activities, including hydrolase, oxidoreductase and transferase, which are associated with CTL functions. Our findings indicate that the biological function and morphology of exosomes secreted by CTLs are influenced by the stimulation CTLs have received. This suggests that a functional, antigen-specific CTL response may boost functions of CTLs against other pathogens through secretion of functional exosomes. This may be a new mechanism for homeostatic regulation in CTL immune responses.
Ten-day exposure to low doses of ultrafine black carbon induces stress responses and reduces the vitality of human and murine pulmonary cell lines
Authors: Salinas M Esther
Published: January 2023

Ultrafine particulates (UFP) are emitted into urban air by combustion processes, commonly via vehicular emissions. These respirable particles can induce a series of immune responses contributing to higher incidences of allergy-related asthma. It is unclear how cells that are in direct contact with these inhaled materials are affected. Our project aims to elucidate the effects of ultrafine carbonaceous particle exposure to human and murine pulmonary cells. We surmise that prolonged treatments of ultrafine black carbon (UBC) directly contribute to lung irritation. In vitro studies were conducted using UBC treatments after a 10-day incubation period. Viability data has shown that the different concentrations of UBC used were mostly non-toxic and generally altered cellular function displaying an inverse dose-dependent manner. Biochemical assays indicated decreased metabolic activity; no significant negative effect on viability was observed in the epithelial cell lines. In the macrophages/monocytes, no damaging physiological activity and no compromising effects on cellular vitality were displayed. The induction of oxidative stress was also assessed using confocal microscopy showing a high number of ROS with increasing UBC concentrations, yet no marked trends were noticed upon conducting mitochondrial de-polarization assays via flow cytometry. This regimen demonstrates that various concentrations of UBC treatments affect mammalian cell function distinctly. In summation, this study suggests that carbonaceous pollutants negatively impact intracellular functionality and overall cellular health. Our findings may potentially provide treatments for allergy-induced asthma upon chronic exposure to ambient nanocarbon.
Gimap5-dependent inactivation of GSK3 is required for CD4 T cell homeostasis and prevention of immune pathology
Authors: Hoebe Kasper , Patterson Andrew , Endale Mehari , Lampe Kristin , Bleesing Jack
Published: January 2023

The GTPase of immunity-associated protein 5 (Gimap5) has been linked with lymphocyte survival, autoimmunity and colitis development. Previous studies indicated that Gimap5-deficient mice exhibit a reduced lymphocyte proliferation and survival. Here we show that Gimap5 is essential for inactivation of Glycogen Synthase Kinase-3β (GSK3β) following T cell activation, a constitutive active Ser/Thr kinase whose activity reduces β-catenin and c-Myc levels thereby maintaining T cell quiescence. Gimap5 regulates the subcellular localization of GSK3β and its phosphorylation at Ser389, which limits DNA damage during T cell proliferation. Importantly, pharmacologic and genetic targeting of GSK3 in mice can override Gimap5-deficiency in CD4+ T cells, preventing exacerbated DNA damage while enhancing T cell proliferation and survival. Additionally, this ameliorates immune-mediated pathology in vivo. Finally, we show that a human patient carrying a GIMAP5 loss-of-function mutation exhibits similar T cell defects and pathologies and exhibit a reduced T cell proliferation and survival associated with increased DNA damage that can be corrected in vitro with GSK3 inhibitors. T cells require a robust clonal expansion during an effective immune response. Given the predominant expression of GIMAP5 in lymphocytes, we propose that the identified pathway presents a novel and critical molecular mechanism required for productive CD4+ T cell responses and immune homeostasis in the periphery.
Lack of polyfunctional cytomegalovirus-specific T cells in hemodialysis patients
Authors: Chiu Yen-Ling , Lay Fang-Yun , Shu Kai-Hsiang , Chou Tzu-Ying , Chia Jean-San , Schneck Jonathan P
Published: January 2023

Polyfunctional T cells are critical for maintaining protection against pathogens. Patients with end-stage renal disease (ESRD) are at increased risks for infectious complications and their T cell immunity against viruses may be impaired. The current study intends to investigate the effects of renal disease on T cell immunity by analyzing T cell differentiation (based on expression of CCR7 and CD45RA) and polyfunctionality response against cytomegalovirus (CMV), an ubiquitous pathogen. Two CMV peptide pools (IE1 and pp65) were used to stimulate PBMCs and four effector functions were measured by multicolor flow cytometry (IL-2, TNFα, IFNγ and CD107a) to identify polyfunctional T cells. 17 healthy individuals, 13 patients with pre-dialysis chronic kidney disease (CKD) and 37 ESRD patients were enrolled in this study. All the donors were seropositive with an average age of 60. Patients with renal disease, especially the ESRD patients, showed increased levels of T cell differentiation, including the decrease in CD4+ and CD8+ TN cells and the increase in the CD4+ and CD8+ TEM and TEMRA cells. While the cellular frequency of CMV-reactive CD4+ and CD8+ T cells were similar among different groups, polyfunctional cell were dramatically reduced in ESRD. Among healthy individuals, CKD and ESRD patients, CD4+ CMV-IE1-reactive polyfunctional cell frequencies were 2.0%, 2.2%, and 0%, respectively (p=0.002). The CD8+ CMV-pp65-reactive polyfunctional cell frequencies were 12.4%, 8.8% and 0.8% in the three groups (p<0.001). Such phenomenon resembles T cell exhaustion observed in HIV infection and further studies should investigate the level of inhibitory receptor expression levels such as PD-1 and CTLA-4 in these patients.
Characterization of keratinocytes isolated from human suction blisters and passaged with Rho kinase inhibitor
Authors: Datta Sandip K , Anderson Erik , Sastalla Inka , Moore Ian N. , Myles Ian Antheni
Published: January 2023

Keratinocytes are the most abundant cell type in the epidermis. They prevent desiccation and provide immunological and barrier mechanisms of defense against potential pathogens such as Staphylococcus aureus and Candida albicans. Study of this important first line of immune defense is hindered by the invasive isolation methods and insufficient techniques for long-term passage of primary keratinocytes in vitro. One method for obtaining primary human keratinocytes is to isolate them from blister roofs induced by negative pressure suction that separates the epidermis from the dermis in vivo in human subjects. This method allows collection of pure epidermal cells without dermal contamination in a minimally invasive manner. However, the method falls short because the isolated keratinocytes differentiate and senesce when cultured in vitro, with the maximum passage number being reported at 5. To address this problem of in vitro cultivation, we present evidence that the Rho kinase (ROCK) inhibitor Y-27632 can be used to effectively increase the proliferative capabilities of keratinocytes isolated using the suction-blister method, similar to what has been previously reported for neonatal foreskin-derived keratinocytes. We also show that upon removal of the ROCK inhibitor the cells retain their ability to stratify into an epithelium as well as produce human beta-defensins upon exposure to IL-17 and interferon-gamma. We expect that when coupled with the minimally invasive and efficient nature of the suction blister technique, our methodology could become a preferred procedure for keratinocyte isolation and cultivation that will allow analysis of keratinocyte function in both healthy volunteers and patients with diseases of interest.
Rapid, Large Scale Experiments to Monitor Day-to-Day Cytokine Secretion, T-Cell Activation and Immunophenotype in T-Cell Activation Protocols
Authors: Duensing Thomas , Liu Zhaoping
Published: January 2023

Monitoring the ex vivo activation of human T lymphocytes is key to developing optimized adoptive cell therapy protocols. We describe here the development of a large scale multiplexed assay using high throughput flow cytometry to daily monitor T-cell activation in PBMCs from multiple donors. On Day 0, PBMCs from multiple donors were profiled to confirm immunophenotype before and after T-cell enrichment with CD3/CD28 magnetic beads. Enriched T cells were cultured in the presence of the beads for 3 days. On Days 1, 2, and 3, samples were removed from the cultures and evaluated using assays that combined cell phenotype measurements with analysis of multiple secreted cytokines. In each well of the assay plates, secreted levels of 11 cytokines and multiple cellular readouts including the presence of cells expressing CD69, CD25 and HLA-DR over the course of a three-day activation protocol were determined. Cell viability and cell number for different subpopulations were also evaluated. Each 96-well plate contained T cells from 12 different donors, in quadruplicate, in two different dilutions. Each plate was read on the iQue® Screener PLUS system in less than 15 minutes. The results indicate time resolved, donor-to-donor differences in activation markers. These large-scale experiments with high throughput flow cytometry provide extensive T-cell activation profiles on multiple donors with rapid assay turnaround time. The use of these assays can provide valuable information for optimizing adoptive cell therapy development and manufacturing protocols.
A simple and integrated workflow for deep proteomic and transcriptomic analysis of sorted cell populations
Authors: Hinerfeld Douglas , Fuhrman KIt , Demirkan Gokhan , DosSantos Gary , Geiss Gary
Published: January 2023

In addition to the long standing efforts to understand and manipulate the immune system in the treatment of autoimmune diseases, the immune system is increasingly becoming a direct target for cancer therapeutics. Recent advances in flow and mass cytometry have greatly expanded the number of immune cell parameters that can be interrogated resulting in an improved understanding of the immune system heterogeneity. These technologies, however, remain limited in the number and types of analytes that can be examined in a single clinical sample. The NanoString nCounter® platform enables the highly multiplexed digital analysis of both RNA and protein from a single biological specimen for multiple research applications. We have recently developed the nCounter® Vantage 3D™ RNA:Protein Immune Cell Profiling Assay for research applications, which interrogates 30 cell surface proteins and 770 immune-related RNAs starting with cells in suspension. Expanding on this, we demonstrate the development of a streamlined workflow that integrates standard immune cell sorting with downstream nCounter® analysis. By co-staining PBMCs with both fluorescently-labeled and DNA barcoded antibodies, CD8+ and CD4+ T cells and CD19+ B cells were isolated followed by analysis of dozens of additional proteins and 770 RNA from each sorted population. Demonstrating the value of this workflow in analyzing potentially rare cell populations, the number of target cells were titrated to determine the sensitivity of the workflow. Without the requirement for additional molecular biology methods, such as RNA purification or sequencing library construction, this method is ideally suited for incorporation into any cell sorting workflow.
Natural killer cell development and maturation is regulated by Sostdc1
Authors: Millan Alberto Jovani , Elizaldi Sonny , Lee Eric , Murugesh Deepa , Loots Gabriela , Manilay Jennifer O
Published: January 2023

Natural killer (NK) cells are specialized lymphocytes with innate ability to eliminate virally infected and cancerous cells. NK cells stochastically express Ly49 activating and inhibitory surface receptors, through mechanisms that are not fully understood. Ly49 expression during NK cell development is regulated by a combination of external and internal factors, such as major histocompatibility complex class I (MHC-I) ligands and cytokines. Global deletion of sclerostin domain containing-1 (Sostdc1), a secreted protein, leads to altered bone homeostasis via dysregulation of Wnt and BMP signaling, which in turn, could result in altered immune cell development in the bone marrow (BM). Using Sostdc1-knockout (KO) mice, we have discovered a novel role for Sostdc1 in NK cell development. Flow cytometric analysis of the Ly49 repertoire in immature (CD27+CD11b-; iNK), transitional (CD27+CD11b+, tNK) and most mature (CD27-CD11b+, mNK) Sostdc1-KO NK cells demonstrated expression differences in Ly49G2, I, H, and D receptors. We also observed a progressive increase in tNK cell numbers and frequencies as the KOs aged from 12 weeks to 30 weeks, indicating a slowing or partial block in NK cell maturation with aging. Reciprocal BM chimera experiments showed that Sostdc1 regulates NK cells through cellintrinsic and cell-extrinsic manners. Consistent with this, Sostdc1 is expressed in the bone periosteum and we discovered Sostdc1 expression in CD45+ NK1.1-CD11b+ BM cells and iNK cells. Taken together, these data support a role for Sostdc1 in the regulation of NK cell differentiation and identify potential NK cell niches. Current studies are investigating whether Sostdc1-KO NK cells respond to stimulation and lyse MHC-I deficient target cells.
Diet-induced weight loss restores T cell number and function in the intestinal epithelium of obese mice
Authors: Jameson Julie M , Miranda Nadia Delgadillo , Limon Natalie , Barba Cindy , Park Christa S
Published: January 2023

Over 30% of adults have a BMI greater than 30 in the United States. Obesity is associated with complications such as type 2 diabetes and chronic non-healing wounds. Our laboratory has demonstrated that obesity and type 2 diabetes negatively impact T cell number, proliferation and cytokine production in the epithelial layer of the skin and the intestine. However, little is known about whether the number and function of these T cells can be restored to normal levels. Here we investigated whether diet-induced weight loss would restore T cells in the small intestine of obese mice. Mice were placed on either 10% kcal Normal Chow Diet (NCD) for 14 weeks, 60% kcal high fat diet (HFD) for 14 weeks, or 60% kcal HFD for 7 weeks followed by a 10% kcal diet for the remaining 7 weeks. The small intestine was examined by snap freezing the tissue, cutting 5um sections, staining the tissue sections for T cells, and examining the sections by immunofluorescent microscopy. Diet-induced weight loss restored T cell numbers to their original levels in the small intestine. In addition, short-term dieting restored the expression of adhesion molecules such as CD103 and chemokine receptors such as CCR9 on the IEL. Previously we have shown that obese mice are more sensitive to DSS-induced colitis. Diet-induced weight loss rescued mice from excessive bleeding and weight loss due to DSS-induced damage. Together this data suggests that while obesity impairs lymphocytes in barrier tissues such as the intestinal epithelium, diet-induced weight loss can restore T cell number and improve the outcome from colitis.
Young serum-derived exosomes potentially attenuate inflammaging via partially rejuvenating immuotolerance (Supported by NIH/NIAID R01AI121147 to D-M. S.)
Authors: Wang Weikan , Wang Liefeng , Oh Jiyoung , Zhuge Qichuan , Su Dongming
Published: January 2023

Aging results in a chronic inflammatory condition, termed inflammaging, establishing a risk for age-related, such as neurocardiovascular, diseases. Therefore, it is of great importance to develop rejuvenating strategies able to attenuate inflammaging as a means to help intervention of age-related diseases. A promising “rejuvenation factor” present in young blood has been found to be able to make aged neuron “younger”. However, the component in the young serum is largely unclear. Herein, we tested rejuvenation in naturally-aged mice with a potential factor – young serum-derived exosomes, based on different spectrums of microRNAs in the young and old exosomes. We found that young murine serum-derived exosomes were able to attenuate inflammaging in both the periphery and the central nerve system of old mice. In addition to senescent cells secreting pro-inflammatory factors, thymic aging-released self-reactive T cell-induced autoimmune predisposition is another potential etiology of inflammaging, we identified that the young serum-derived exosome-attenuating inflammaging was partially attributed to rejuvenation of thymic aging, characterized by reversed thymic involution and improved function in negative selection. Our pilot work provides a clue of the rejuvenation factor in the young serum, which holds great promise toward the development of novel therapeutics to reduce morbidity and mortality caused by age-related diseases.
Reactive oxygen species regulate the inflammatory function of NKT cells through Promyelocytic Leukaemia Zinc Finger (PLZF) protein
Authors: Kim Yeunghyen , Pyaram Kalyani , Kumar Ajay , Chang Cheong-Hee
Published: January 2023

Cellular metabolism controlled by the signaling pathways has a crucial role in dictating the outcome of T cell activation and effector function. Reactive oxygen species (ROS) are byproducts of aerobic metabolism and contribute to both physiological and pathological conditions as a second messenger. In T cells, ROS levels increase upon activation and antigen specific responses of T cells are compromised in the absence of ROS production by mitochondria. However, it is not well understood how NKT cells’ functions are controlled by cell metabolism. In the current study, we investigated the role of ROS in NKT cell function. We found that ROS levels were similar among CD4, CD8 and NKT cell subsets in the thymus but NKT cells showed dramatically increased ROS in the spleen and liver but not in adipose tissues. ROS in the peripheral NKT cells were primarily produced by NADPH oxidases not mitochondria. Accordingly, ROS high NKT cells were susceptible to oxidative stress and underwent apoptotic cell death. Furthermore, ROS play an important role regulating the inflammatory function of NKT cells because antioxidant treatment of NKT cells showed reduced frequencies of IFN-γ+ and IL-17+ cells. In line with this, freshly isolated ROS-high NKT cells had more NKT1 and NKT17 cells but less NKT2 than ROS-low cells. Importantly, these characteristics of NKT cells are regulated by PLZF (Promyelocytic Leukaemia Zinc Finger) protein as evidenced by reduced ROS in NKT cells from adipose tissues that do not express PLZF and also those from PLZF haplodeficient mice. Conversely, ROS were highly elevated in CD4 T cells from mice ectopically expressing PLZF. Together, our study revealed for the first time that ROS regulate NKT cell functions through PLZF.
Complement component C1q regulates AMPK and ERK1/2 activation in mouse and human macrophages
Authors: Willmann Emily A , Pandurovic Vesna , Bohlson Suzanne
Published: January 2023

C1q is the recognition component of the classical complement pathway and a soluble pattern recognition receptor that regulates myeloid cell activation independent of the complement cascades. C1q enhances phagocytosis of antibody opsonized targets and apoptotic cells, and regulates cytokine production in macrophages. C1q deficiency results in autoimmunity, consistent with its function in regulating macrophage activation. The signaling mechanisms leading to C1q-dependent macrophage activation are not fully understood. To investigate the mechanism by which C1q regulates macrophage activation, we utilized a phospho-kinase array to identify targets within the signaling pathway(s). The array identified several targets that were regulated by C1q including AMP-dependent protein kinase α-1 (AMPK) and ERK1/2. Short term stimulation (30 min) with C1q and subsequent addition of immune complexes resulted in enhanced ERK phosphorylation in C1q-treated THP-1 cells and mouse bone marrow derived macrophages (BMDM). These same conditions stimulate enhanced phagocytosis of targets suboptimally opsonized with IgG. Long term stimulation (18 hr) of BMDM with C1q under conditions that lead to enhanced engulfment of apoptotic cells and dampening of proinflammatory TNFα production led to enhanced AMPK activation and inhibition of ERK, consistent with C1q enhancing efferocytosis and dampening TNF production. Silencing AMPK resulted in inhibition of C1q-dependent efferocytosis, however AICAR, the AMPK agonist, was not sufficient for restoring C1q-dependent signaling. These data identify new targets of C1q-dependent signaling which may be important in developing strategies to prevent autoimmune and/or inflammatory diseases.
A tissue-resident macrophage specific coinhibitory molecule promotes regulatory T cell differentiation and stability
Authors: Fu Wenxian , Yuan Xiaomei , Yang Bi-Huei , Dong Yi
Published: January 2023

CD4+Foxp3+ regulatory T (Treg) cells play a central role in the prevention of lethal autoimmunity and excessive inflammation. In contrast to their counterparts in lymphoid organs, Treg cells in non-lymphoid tissues acquire tissue-specific functional properties. While Treg tissue-tropisms are increasingly recognized, it remains poorly understood how tissue microenvironment influences Treg development, function and stability. Complement receptor of immunoglobulin family (CRIg) is a recently identified B7/CD28 family member. CRIg is exclusively expressed in tissue-resident macrophages. Earlier studies have found that CRIg functions as a coinhibitory molecule to suppress T cell proliferation and cytokine production. We here report a completely novel mechanism by which CRIg regulates T cell biology. We find that CRIg promotes the development of TGF-b induced regulatory T (iTreg) cells. More importantly, CRIg stabilizes the expression of Foxp3 in Treg cells by enhancing their responsiveness to IL-2. In vivo modulation of CRIg increases Treg abundancy and restores immune tolerance in autoimmune condition. In summary, these data shed new light on how tissue-resident macrophages impact tissue homeostasis by regulating the development and stability of Treg cells. CRIg may represent a tissue-specific immunomodulation to promote immune tolerance.
ICOS signaling suppresses an excessive T cell response in the CNS during chronic T. gondii infection
Authors: O’Brien Carleigh , Harris Tajie H
Published: January 2023

While the importance of Tregs in limiting T cell responses during the generation of an effector immune response to pathogens has been shown, the exact mechanisms by which Tregs limit immunopathogenic T cell responses during chronic inflammation of the brain remain unknown. We find that Tregs are recruited to the brain during toxoplasmic encephalitis, where a strong effector T cell response is necessary to limit replication of reactivated parasite. Inducible T cell costimulator (ICOS) has been shown to be both critical for developing optimal effector T cell responses to pathogens, as well as necessary for Treg survival in a homeostatic state. However, the role of ICOS signaling during the maintenance of long-term immune responses to chronic inflammation remains poorly understood. We find that Tregs recruited to the CNS during chronic infection express higher levels of ICOS than effector T cells, and that blocking ICOS signaling during chronic infection unexpectedly leads to increased T cell responses and immune-mediated pathology in the brain. This immunopathogenic effect is limited to the CNS, where the increased effector T cell response seen with ICOS-ligand blockade is associated with higher CD25 and Bcl-2 expression, suggesting a lack of ICOS signaling enhances effector T cell survival in the brain during chronic inflammation. Surprisingly, no changes in the costimulatory properties of APCs in the brain have been observed, suggesting a lack of regulation within the T cell population without ICOS costimulation at sites of inflammation. The excessive T cell response seen in the absence of ICOS signaling suggests an important role for ICOS-ICOSL costimulation in maintaining local immune suppression in the CNS during chronic infection.
MicroRNAs of the miR-23~27~24 family coordinately regulate T follicular helper cell responses during allergic reaction and viral infection
Authors: Wu Cheng-Jang , Cho Sunglim , Cruz Leilani Ong , Lin Ling-Li , Chen Mei-Chi , Lu Li-Fan
Published: January 2023

T Follicular helper (Tfh) cells are essential for forming geminal centers (GCs) and promoting protective humoral immunity. On the other hand, aberrant Tfh cell responses could also lead to the production of autoantibodies and the development of autoimmunity. To date, microRNAs (miRNAs) have emerged as key molecular players in regulating Tfh cell differentiation and homeostasis. Here, by using both lost-of-function and gain-of-function approaches, we show that loss of miR-23~27~24 clusters in T cells resulted in elevated frequencies of Tfh and GC B cells upon allergen challenge as well as during viral infection whereas T cell-specific overexpression of this miRNA family led to reduced Tfh cell responses. Interestingly, unlike our previously reported findings which showed that individual members of this miRNA family do not always work with each other in regulating other T cell lineages, we found that the entire miR-23~27~24 family coordinately control Tfh cell responses through targeting a network of genes that are critical for Tfh cell differentiation and function. Collectively, our results establish miR-23~27~24 clusters as important regulators in maintaining optimal Tfh cell responses and the resultant humoral immunity.
Identification of a role for Casz1 in the control of T helper differentiation and inflammation
Authors: Pandiyan Pushpa , Bhaskaran Natarajan , Thiele Carol J , Weinberg Aaron
Published: January 2023

While T helper (Th) cells constitute a critical arm of adaptive immune system and are important for host defense, unregulated expansion and imbalance in Th effector subsets contribute to inflammatory disorders. Here we show that Casz1, whose function is previously unknown in CD4+ T cells, determines the balance between various Th subsets. As systemic knockout mice are embryonically lethal, we generated conditional CD4 Casz1 knockout mice. Conditional deletion of Casz1 in CD4+ T cells significantly inhibits Th17 cell differentiation, but promotes Th1 differentiation. Loss of Casz1 in CD4+ T cells lowers susceptibility to experimental autoimmune encephalomyelitis, consistent with the reduced frequency of Th17 cells, despite an increase in Th1 cells. These results underscore the critical role of Casz1 in determining Th17 lineage differentiation in vivo. Transcriptome analyses of Casz1 deficient CD4+ T cells show a signature consistent with defective Th17 differentiation but enhanced Th1 differentiation. Taken together, these data reveal Casz1 as a new T helper cell regulator having important clinical implications for autoimmune inflammation.
O-GlcNAc Regulates CD4+ T Cell Differentiation
Authors: Machacek Miranda E , Li Jibiao , Li Tiangang , Slawson Chad , Fields Patrick
Published: January 2023

Chronic inflammation is a feature of obesity and enhances the risk of heart disease, cancer, and diabetes. Specifically, pro-inflammatory TH1 and TH17 CD4+ effector T cells are increased in metabolic diseases. However, a clear molecular mechanism linking metabolic changes with pro-inflammatory T cells is lacking. We hypothesize that elevated levels of O-linked β-N-acetylglucosamine (O-GlcNAc), a post-translational modification (PTM) of nuclear and cytoplasmic proteins, promotes pro-inflammatory CD4+ T cell differentiation. Since production of O-GlcNAc involves input from multiple metabolic pathways, the PTM acts as a nutrient sensor. To investigate the role of O-GlcNAc in a setting of metabolic disease, we analyzed O-GlcNAc levels in CD4+ T cells in mice fed a high fat diet. CD4+ T cells from obese mice have elevated O-GlcNAc levels and are primed to secrete more IL-17A, the eponymous TH17 cytokine, and IFNγ, the signature TH1 cytokine. We also cultured mouse splenic CD4+ T cells with Thiamet-G (TMG), a selective inhibitor of the enzyme that removes O-GlcNAc. We find a significant increase in protein and transcript levels of IL-17A and IFNγ in TMG treated cells. Acetyl CoA carboxylase (ACC1), the rate limiting enzyme in fatty acid synthesis, is known to enhance generation of TH17 cells by producing ligands that increase the activity of RORγt, the transcription factor defining the TH17 lineage. We find that ACC1 is O-GlcNAcylated in CD4+ T cells. Collectively, our data suggest that elevated O-GlcNAc levels prime TH17 CD4+ T cell differentiation by altering ACC1 activity. Further study into how O-GlcNAcylation promotes pro-inflammatory T cell differentiation will provide insight into how nutritional excess exacerbates inflammation.
Exogenous truncated IK protein ameliorates inflammatory arthritis.
Authors: Jung SeoYeon , Choi Seulgi , Park Hye-Lim , Nam Jae-Hwan
Published: January 2023

The truncated IK (tIK) cytokine is known for its ability to downregulate the major histocompatibility complex class II on antigen-presenting cells and to regulate immune activation in inflammatory diseases by suppressing differentiation of pathogenic T cells and activation of macrophage. Because of these immune regulatory functions, previous research has proposed tIK as a new candidate for autoimmune disease therapy such as that for rheumatoid arthritis (RA). However, its immunological mechanism is still not identified clearly. In this study, we showed that exogenous tIK protein produced by the insect expression system suppressed the differentiation of TH1 and TH17 cells, which are key factors of inflammatory arthritis. We also showed that tIK protein reduced symptoms of the disease in the collagen antibody-induced arthritis (CAIA) mouse model. In addition, to investigate the receptor and signal transducers that directly interact with tIK and/or the intracellular molecules as a signal pathway, we performed a protein–protein interaction array. According to protein array data, FAM104B, CUTA, GSN, YEATS4 and some other proteins might interact with tIK protein. Among them some proteins may serve as the negative regulators of NF-kappaB transcription factor. Therefore, we suggest that tIK protein has the potential to act as a new therapeutic agent for RA patients, because it has a different mode of action compared with the currently-used biologics for RA, such as monoclonal antibody drugs.
Mesenchymal stem cells inhibit RANK-RANKL interactions between osteoclasts and Th17 cells via osteoprotegerin activity.
Authors: Cho Kyung-Ah , Park Minhwa , Kim Yu-Hee , Ryu Kyung-Ha , Woo So-Youn
Published: January 2023

Th17 cells play a critical role in several autoimmune diseases, including psoriasis and psoriatic arthritis (PsA). Psoriasis is a chronic inflammatory skin disease associated with systemic inflammation and comorbidities, such as PsA. PsA develops in nearly 70% of patients with psoriasis, and bone erosion is a hallmark of the disease. Thus far, the effect of Th17 cells on osteoclastogenesis via direct cell-to-cell interactions is less understood. In this study, we observed that Th17 cells directly promote osteoclast differentiation and maturation via expression of receptor activator of nuclear factor-k β ligand (RANKL) in vitro. We investigated the impact of conditioned medium obtained from human palatine tonsil-derived mesenchymal stem cells (T-CM) on the interactions between osteoclasts and Th17 cells. T-CM effectively blunted the RANK-RANKL interaction between the osteoclast precursor cell line RAW 264.7 and Th17 cells via osteoprotegerin (OPG) activity. The frequency of tartrate-resistant acid phosphatase (TRAP)-positive cells in the bone marrow of an imiquimod (IMQ)-induced psoriasis mouse model was decreased following T-CM injection. Therefore, our data provide novel insight into the therapeutic potential of tonsil-derived mesenchymal stem cell-mediated therapy (via OPG production) for the treatment of pathophysiologic processes that occur in joints or bones under chronic inflammatory conditions such as psoriasis.
Key serum cytokine markers for evaluating the efficacy of vaccine therapy in autoimmune models of rheumatoid arthritis
Authors: Zimmerman Daniel H , Rosenthal Kenneth , Carambula Roy , Cress Steve , Markovics Adrienn , Glant Tibor , Mikecz Katalin
Published: January 2023

Background

Pro-inflammatory T helper (Th)17 or Th1 cell responses (IL17, IFNγ, TNFα) drive autoimmune pathology in rheumatoid arthritis (RA) at the expense of anti-inflammatory/regulatory (IL4, IL10, TGFβ) T cell responses. Ligand Epitope Antigen Presenting System (LEAPS) vaccines, composed of cell-binding and immunodominant peptides, can down-modulate Th17 or Th1 activity and thus have the potential to treat ongoing RA. Choice of appropriate treatment requires knowledge of key cytokines driving disease as well as responses to therapy.

Experimental design

Serum cytokines were assayed in mouse models of RA including proteoglycan-induced arthritis (PGIA) recombinant proteoglycan G1 domain-induced arthritis (GIA) and collagen-induced arthritis (CIA) after arthritic mice had been treated with LEAPS vaccines. Serum cytokine levels and ratios of anti-inflammatory to pro-inflammatory cytokines were correlated with visual and histopathologic scores of disease.

Results

Serum levels of pro-inflammatory cytokines were elevated in control mice with either PGIA, GIA, or CIA. Reduced disease progression in mice treated with LEAPS vaccines was accompanied by decreased pro-inflammatory cytokine levels and increased ratios of anti-inflammmatory/regulatory to pro-inflammatory cytokines.

Conclusion

Serum concentrations and ratios of key disease-related cytokines can predict therapeutic efficacy in different animal models of RA. Successful vaccine therapy is associated with ratios shifted in favor of anti-inflammatory/regulatory cytokines. The ability to predict cytokine responses to therapy will allow better design or choice of appropriate immunomodulatory LEAPS vaccines and other therapies in RA.
TAK-079 is a high affinity monoclonal antibody that effectively mediates CD38+ cell depletion
Authors: Smithson Glennda , Zalevsky Jonathan , Korver Wouter , Roepcke Stefan , Dahl Martin , Zhao Lin , Yuan Josh , McLean Lachy , Elias Kathleen
Published: January 2023

TAK-079 is a fully human, non-agonistic, IgG1 cell-depleting monoclonal antibody that binds human CD38 with high affinity (KD 1.2 nM, KinExA) and cross reacts with cynomolgus monkey CD38. CD38 is a cADPR hydrolase expressed on human plasmablasts, plasma cells, NK cells and activated T and B cells, but not on mature platelets or red blood cells, based on TAK-079 binding. In patients with rheumatoid arthritis (RA) and systemic lupus erythematousus (SLE), plasma cells, as well as activated B and T cells may be important contributors to disease. Unlike other B cell-selective therapies which target CD20 and do not directly deplete plasmablasts, which are CD20low/negative, CD38 is expressed at high levels on plasmablasts and plasma cells making these cells a direct target of TAK-079. In vitro studies with human blood cells and cell lines showed that binding of TAK-079 to CD38 did not result in PBMC cytokine activation demonstrating that TAK-079 is not an agonist. Rather, it mediated depletion of human B lineage cell lines by ADCC and CDC and in most cases cell lines with increased CD38 expression were more susceptible to cell lysis. This is consistent with findings in healthy cynomolgus monkeys where the efficiency of depletion correlated with level of CD38 expression and TAK-079 dose level. NK cells, which express high levels of CD38, were depleted to a greater extent than CD20+ B cells and CD3+ T cells, which express less CD38. In vivo, TAK-079 potently suppressed the human B cell recall responses to antigen in a mouse adoptive transfer model. Together these data support the further investigation of TAK-079 in autoimmune diseases.