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1.
Sci Rep ; 14(1): 12292, 2024 05 29.
Article in English | MEDLINE | ID: mdl-38811654

ABSTRACT

The UV-mediated peptide exchange has enabled the generation of multiple different MHC multimer specificities in parallel, surpassing tedious individual refolding of MHC molecules with peptide ligands. Murine models are acknowledged as an effective tool for preclinical research to advance our understanding of immunological mechanisms, with the potential translatability of key learnings from mouse models to the clinic. The common inbred mouse strain BALB/c is frequently used in immunological research. However, for the BALB/c histocompatibility (H)-2 alleles availability of conditional ligand has been limited. To overcome this challenge, we design and experimentally validate conditional ligands restricted to murine MHC class I alleles H2Dd and H2Kd. In addition, we demonstrate the ability of the three H2d molecules and two additional C57BL/6 H2b molecules folded in-house with conditional ligands to generate fluorescently labeled peptide-H2 tetramers that allow staining of antigen-specific CD8+ T cells in splenocyte samples. Finally, we generate large peptide-H-2 multimer libraries with a DNA-barcode labeling system for high-throughput interrogation of CD8+ T cell specificity in murine splenocyte samples. Consequently, the described techniques will contribute to our understanding of the antigen-specific CD8+ T cell repertoire in murine preclinical models of various diseases.


Subject(s)
CD8-Positive T-Lymphocytes , Peptides , Animals , Ligands , Mice , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Peptides/immunology , Peptides/chemistry , Mice, Inbred C57BL , H-2 Antigens/immunology , H-2 Antigens/metabolism , H-2 Antigens/genetics , Mice, Inbred BALB C
2.
Transplantation ; 108(6): 1357-1367, 2024 Jun 01.
Article in English | MEDLINE | ID: mdl-38361235

ABSTRACT

BACKGROUND: The formation of anti-major histocompatibility complex (MHC) antibodies is a significant barrier for many patients awaiting organ transplantation. Patients with preformed anti-MHC antibodies have limited options for suitable donors, and the formation of donor-specific anti-MHC antibodies after transplantation is a harbinger of graft rejection. Despite the recognized importance of anti-MHC antibodies, the mechanisms responsible for the differentiation of B cells after exposure to allogeneic antigens are poorly understood. METHODS: To evaluate the differentiation of B cells in response to allogeneic antigen, we used a model of H-2 b C57Bl/6 sensitization with H-2 d antigen. We used a class I MHC tetramer-based approach to identify allogeneic B cells and flow cytometric crossmatch to identify allogeneic IgM and IgG. RESULTS: We found that although the formation of anti-H-2 d IgG was robust, few class-switched B cells and germinal center B cells were formed. Antigen-specific B cells did not express classical memory B-cell markers after sensitization but had an IgM + CD21 + marginal zone B-cell phenotype. The frequency of marginal zone B cells increased after sensitization. Depletion of marginal zone B cells before sensitization or skin grafting resulted in a significant diminution of anti-H-2 d IgG and fewer germinal center B cells. Adoptive transfer experiments revealed that marginal zone B cells more efficiently differentiated into germinal center B cells and anti-donor IgG-producing cells than follicular B cells. CONCLUSIONS: These results demonstrate an important role for marginal zone B cells as a reservoir of alloreactive B cells that are activated by allogeneic antigens.


Subject(s)
B-Lymphocytes , Immunoglobulin G , Isoantibodies , Mice, Inbred C57BL , Skin Transplantation , Animals , Immunoglobulin G/immunology , B-Lymphocytes/immunology , Isoantibodies/immunology , Isoantibodies/blood , Cell Differentiation/immunology , Mice , H-2 Antigens/immunology , Graft Rejection/immunology , Graft Rejection/prevention & control , Transplantation, Homologous , Immunoglobulin M/immunology , Phenotype , Germinal Center/immunology
3.
Biochim Biophys Acta Mol Basis Dis ; 1870(2): 166988, 2024 02.
Article in English | MEDLINE | ID: mdl-38070583

ABSTRACT

Psoriasis is a multifaceted chronic inflammatory skin disease; however, its underlying molecular mechanisms remain unclear. In this study, we explored the role of fucosylation in psoriasis using an imiquimod-induced psoriasis-like mouse model. ABH antigen and fucosyltransferase 1 (Fut1) expression was reduced in the granular layer of lesional skin of patients with psoriasis. In particular, the blood group H antigen type 2 (H2 antigen)-a precursor of blood group A and B antigens-and FUT1 were highly expressed throughout the spinous layer in both patients with psoriasis and the skin of imiquimod-treated mice. Upon the application of imiquimod, Fut1-deficient mice, which lacked the H2 antigen, exhibited higher clinical scores based on erythema, induration, and scaling than those of wild-type mice. Imiquimod-treated Fut1-deficient mice displayed increased skin thickness, trans-epidermal water loss, and Gr-1+ cell infiltration compared with wild-type mice. Notably, the levels of CXCL1 protein and mRNA were significantly higher in Fut1-deficient mice than those in wild-type mice; however, there were no significant differences in other psoriasis-related markers, such as IL-1ß, IL-6, IL-17A, and IL-23. Fut1-deficient primary keratinocytes treated with IL-17A also showed a significant increase in both mRNA and protein levels of CXCL1 compared with IL-17A-treated wild-type primary keratinocytes. Further mechanistic studies revealed that this increased Cxcl1 mRNA in Fut1-deficient keratinocytes was caused by enhanced Cxcl1 mRNA stabilization. In summary, our findings indicated that fucosylation, which is essential for ABH antigen synthesis in humans, plays a protective role in psoriasis-like skin inflammation and is a potential therapeutic target for psoriasis.


Subject(s)
Blood Group Antigens , Psoriasis , Humans , Animals , Mice , Imiquimod/adverse effects , Interleukin-17/genetics , Interleukin-17/metabolism , H-2 Antigens/adverse effects , Psoriasis/chemically induced , Psoriasis/genetics , Inflammation/chemically induced , RNA, Messenger/genetics , RNA, Messenger/metabolism , Blood Group Antigens/adverse effects , Chemokine CXCL1/genetics
4.
Proc Natl Acad Sci U S A ; 120(43): e2304689120, 2023 10 24.
Article in English | MEDLINE | ID: mdl-37856544

ABSTRACT

The importance of classical CD8+ T cells in tumor eradication is well acknowledged. However, the anti-tumor activity of MHC (major histocompatibility complex) Ib-restricted CD8+ T (Ib-CD8+ T) cells remains obscure. Here, we show that CX3CR1-expressing Ib-CD8+ T cells (Ib-restricted CD8+ T cells) highly express cytotoxic factors, austerely resist exhaustion, and effectively eliminate various tumors. These Ib-CD8+ T cells can be primed by MHC Ia (MHC class Ia molecules) expressed on various cell types for optimal activation in a Tbet-dependent manner. Importantly, MHC Ia does not allogeneically activate Ib-CD8+ T cells, rather, sensitizes these cells for T cell receptor activation. Such effects were observed when MHC Ia+ cells were administered to tumor-bearing Kb-/-Db-/-mice. A similar population of tumoricidal CX3CR1+CD8+ T cells was identified in wild-type mice and melanoma patients. Adoptive transfer of Ib-CD8+ T cells to wild-type mice inhibited tumor progression without damaging normal tissues. Taken together, we demonstrate that MHC class Ia can prime Ib-CD8+ T cells for robust tumoricidal activities.


Subject(s)
CD8-Positive T-Lymphocytes , Histocompatibility Antigens Class I , Humans , Mice , Animals , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/metabolism , H-2 Antigens , Histocompatibility Antigens/metabolism , Mice, Inbred C57BL
5.
Front Immunol ; 13: 868496, 2022.
Article in English | MEDLINE | ID: mdl-35720315

ABSTRACT

Natural killer (NK) cell responses depend on the balance of signals from inhibitory and activating receptors. However, how the integration of antagonistic signals occurs upon NK cell-target cell interaction is not fully understood. Here we provide evidence that NK cell inhibition via the inhibitory receptor Ly49A is dependent on its relative colocalization at the nanometer scale with the activating receptor NKG2D upon immune synapse (IS) formation. NKG2D and Ly49A signal integration and colocalization were studied using NKG2D-GFP and Ly49A-RFP-expressing primary NK cells, forming ISs with NIH3T3 target cells, with or without the expression of single-chain trimer (SCT) H2-Dd and an extended form of SCT H2-Dd-CD4 MHC-I molecules. Nanoscale colocalization was assessed by Förster resonance energy transfer between NKG2D-GFP and Ly49A-RFP and measured for each synapse. In the presence of their respective cognate ligands, NKG2D and Ly49A colocalize at the nanometer scale, leading to NK cell inhibition. However, increasing the size of the Ly49A ligand reduced the nanoscale colocalization with NKG2D, consequently impairing Ly49A-mediated inhibition. Thus, our data shows that NK cell signal integration is critically dependent on the dimensions of NK cell ligand-receptor pairs by affecting their relative nanometer-scale colocalization at the IS. Our results together suggest that the balance of NK cell signals and NK cell responses is determined by the relative nanoscale colocalization of activating and inhibitory receptors in the immune synapse.


Subject(s)
NK Cell Lectin-Like Receptor Subfamily A , NK Cell Lectin-Like Receptor Subfamily K , Animals , Carrier Proteins/metabolism , H-2 Antigens , Histocompatibility Antigen H-2D/metabolism , Killer Cells, Natural , Lectins, C-Type/metabolism , Ligands , Mice , NIH 3T3 Cells , NK Cell Lectin-Like Receptor Subfamily A/metabolism , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Receptors, NK Cell Lectin-Like/metabolism , Receptors, Natural Killer Cell/metabolism
6.
Respir Res ; 23(1): 44, 2022 Mar 03.
Article in English | MEDLINE | ID: mdl-35241086

ABSTRACT

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is among the top 5 causes of mortality in the world and can develop as a consequence of genetic and/or environmental factors. Current efforts are focused on identifying early life insults and how these contribute to COPD development. In line with this, our study focuses on the influence of early life nicotine exposure and its potential impact on (a) lung pulmonary functions, and (b) elastase-induced emphysema in adulthood. METHODS: To address this hypothesis, we developed a model of 2 hits, delivered at different time points: mouse pups were first exposed to nicotine/placebo in utero and during lactation, and then subsequently received elastase/placebo at the age of 11 weeks. The effect of nicotine pretreatment and elastase instillation was assessed by (a) measurement of pulmonary function at post-elastase day (ped) 21, and (b) transcriptomic profiling at ped3 and 21, and complementary protein determination. Statistical significance was determined by 3- and 2-way ANOVA for pulmonary functions, and RNAseq results were analyzed using the R project. RESULTS: We did not observe any impact of nicotine pre- and early post-natal exposure compared to control samples on lung pulmonary functions in adulthood, as measured by FLEXIVENT technology. After elastase instillation, substantial lung damage was detected by x-ray tomography and was accompanied by loss in body weight at ped3 as well as an increase in cell numbers, inflammatory markers in BAL and lung volume at ped21. Lung functions showed a decrease in elastance and an increase in deep inflation volume and pressure volume (pv) loop area in animals with emphysema at ped21. Nicotine had no effect on elastance and deep inflation volume, but did affect the pv loop area in animals with emphysema at ped21. Extensive transcriptomic changes were induced by elastase at ped3 both in the nicotine-pretreated and the control samples, with several pathways common to both groups, such as for cell cycle, DNA adhesion and DNA damage. Nicotine pretreatment affected the number of lymphocytes present in BAL after elastase instillation and some of the complement pathway related proteins, arguing for a slight modification of the immune response, as well as changes related to general body metabolism. The majority of elastase-induced transcriptomic changes detected at ped3 had disappeared at ped21. In addition, transcriptomic profiling singled out a common gene pool that was independently activated by nicotine and elastase. CONCLUSIONS: Our study reports a broad spectrum of transient transcriptomic changes in mouse emphysema and identifies nicotine as influencing the emphysema-associated immune system response.


Subject(s)
Gene Expression Regulation , Histocompatibility Antigens Class I/genetics , Life Expectancy , Nicotine/adverse effects , Pulmonary Emphysema/genetics , RNA/genetics , Animals , Bronchoalveolar Lavage Fluid/cytology , Cells, Cultured , Disease Models, Animal , H-2 Antigens , Histocompatibility Antigens Class I/biosynthesis , Mice , Mice, Inbred C57BL , Pancreatic Elastase/toxicity , Pulmonary Emphysema/chemically induced , Pulmonary Emphysema/metabolism
7.
Front Immunol ; 12: 748679, 2021.
Article in English | MEDLINE | ID: mdl-34721418

ABSTRACT

To circumvent the limitations of available preclinical models for the study of type 1 diabetes (T1D), we developed a new humanized model, the YES-RIP-hB7.1 mouse. This mouse is deficient of murine major histocompatibility complex class I and class II, the murine insulin genes, and expresses as transgenes the HLA-A*02:01 allele, the diabetes high-susceptibility HLA-DQ8A and B alleles, the human insulin gene, and the human co-stimulatory molecule B7.1 in insulin-secreting cells. It develops spontaneous T1D along with CD4+ and CD8+ T-cell responses to human preproinsulin epitopes. Most of the responses identified in these mice were validated in T1D patients. This model is amenable to characterization of hPPI-specific epitopes involved in T1D and to the identification of factors that may trigger autoimmune response to insulin-secreting cells in human T1D. It will allow evaluating peptide-based immunotherapy that may directly apply to T1D in human and complete preclinical model availability to address the issue of clinical heterogeneity of human disease.


Subject(s)
B7-1 Antigen/genetics , Diabetes Mellitus, Type 1/immunology , HLA-DQ Antigens/genetics , Insulin/genetics , Adolescent , Adult , Aged , Amino Acid Sequence , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cytotoxicity, Immunologic , Diabetes Mellitus, Type 1/genetics , Disease Models, Animal , Female , H-2 Antigens/genetics , HLA-A2 Antigen/genetics , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Knockout , Mice, Transgenic , Middle Aged , Young Adult
8.
Front Immunol ; 12: 715893, 2021.
Article in English | MEDLINE | ID: mdl-34594330

ABSTRACT

Allogeneic stem cell transplantation (alloSCT) is a curative therapy for hematopoietic malignancies. The therapeutic effect relies on donor T cells and NK cells to recognize and eliminate malignant cells, known as the graft-versus-leukemia (GVL) effect. However, off target immune pathology, known as graft-versus-host disease (GVHD) remains a major complication of alloSCT that limits the broad application of this therapy. The presentation of recipient-origin alloantigen to donor T cells is the primary process initiating GVHD and GVL. Therefore, the understanding of spatial and temporal characteristics of alloantigen presentation is pivotal to attempts to separate beneficial GVL effects from detrimental GVHD. In this review, we discuss mouse models and the tools therein, that permit the quantification of alloantigen presentation after alloSCT.


Subject(s)
Antigen Presentation/immunology , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/immunology , Transplantation Immunology , Animals , Graft vs Host Disease/etiology , Graft vs Leukemia Effect/immunology , H-2 Antigens/immunology , Hematopoietic Stem Cell Transplantation/adverse effects , Hematopoietic Stem Cell Transplantation/methods , Isoantigens/immunology , Mice , Minor Histocompatibility Antigens/immunology , Molecular Mimicry/immunology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Transplantation, Homologous
9.
J Biol Chem ; 297(4): 101141, 2021 10.
Article in English | MEDLINE | ID: mdl-34478713

ABSTRACT

The CD8αß heterodimer plays a crucial role in the stabilization between major histocompatibility complex class I molecules (MHC-I) and the T cell receptor (TCR). The interaction between CD8 and MHC-I can be regulated by posttranslational modifications, which are proposed to play an important role in the development of CD8 T cells. One modification that has been proposed to control CD8 coreceptor function is ribosylation. Utilizing NAD+, the ecto-enzyme adenosine diphosphate (ADP) ribosyl transferase 2.2 (ART2.2) catalyzes the addition of ADP-ribosyl groups onto arginine residues of CD8α or ß chains and alters the interaction between the MHC and TCR complexes. To date, only interactions between modified CD8 and classical MHC-I (MHC-Ia), have been investigated and the interaction with non-classical MHC (MHC-Ib) has not been explored. Here, we show that ADP-ribosylation of CD8 facilitates the binding of the liver-restricted nonclassical MHC, H2-Q10, independent of the associated TCR or presented peptide, and propose that this highly regulated binding imposes an additional inhibitory leash on the activation of CD8-expressing cells in the presence of NAD+. These findings highlight additional important roles for nonclassical MHC-I in the regulation of immune responses.


Subject(s)
ADP-Ribosylation/immunology , CD8 Antigens/immunology , CD8-Positive T-Lymphocytes/immunology , H-2 Antigens/immunology , Protein Multimerization/immunology , ADP Ribose Transferases/genetics , ADP Ribose Transferases/immunology , ADP-Ribosylation/genetics , Animals , CD8 Antigens/genetics , H-2 Antigens/genetics , Liver/immunology , Mice , Mice, Knockout , Protein Multimerization/genetics , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology
10.
Front Immunol ; 12: 726421, 2021.
Article in English | MEDLINE | ID: mdl-34526998

ABSTRACT

CD8 T cell infiltration of the central nervous system (CNS) is necessary for host protection but contributes to neuropathology. Antigen presenting cells (APCs) situated at CNS borders are thought to mediate T cell entry into the parenchyma during neuroinflammation. The identity of the CNS-resident APC that presents antigen via major histocompatibility complex (MHC) class I to CD8 T cells is unknown. Herein, we characterize MHC class I expression in the naïve and virally infected brain and identify microglia and macrophages (CNS-myeloid cells) as APCs that upregulate H-2Kb and H-2Db upon infection. Conditional ablation of H-2Kb and H-2Db from CNS-myeloid cells allowed us to determine that antigen presentation via H-2Db, but not H-2Kb, was required for CNS immune infiltration during Theiler's murine encephalomyelitis virus (TMEV) infection and drives brain atrophy as a consequence of infection. These results demonstrate that CNS-myeloid cells are key APCs mediating CD8 T cell brain infiltration.


Subject(s)
Antigen-Presenting Cells/pathology , Brain Diseases/virology , Brain/pathology , H-2 Antigens/immunology , Theilovirus/immunology , Animals , Antigen Presentation , Antigen-Presenting Cells/virology , Atrophy , Brain/immunology , Brain/virology , Brain Diseases/immunology , CD8-Positive T-Lymphocytes/immunology , Female , Macrophages/pathology , Macrophages/virology , Magnetic Resonance Imaging , Male , Mice , Mice, Inbred C57BL , Microglia/pathology , Microglia/virology
11.
Int Immunopharmacol ; 100: 108120, 2021 Nov.
Article in English | MEDLINE | ID: mdl-34537480

ABSTRACT

AIMS: The objective of this investigation is to evaluate the mechanisms Anisakis simplex employs to modify its host immune system, regarding the larval antigens interactions with Toll-Like-Receptors (TLRs). METHODS AND RESULTS: In a previous study, we described that the stimulation of bone marrow derived dendritic cells (BMDCs) with A. simplex larval antigens drive an acute inflammatory response in BALB/c mice, but a more discrete and longer response in C57BL/6J. Moreover, when A. simplex larval antigens were combined with TLR agonists (TLR 1/2-9), they modified mainly TLR2, TLR4 and TLR9 agonists responses in both mice strains, and also TLR3, TLR5 and TLR7 in BALB/c. Antigen-presenting ability was analyzed by the detection of CD11c + cells expressing surface markers (CD80-86, MHC I-II), intracellular cytokines (IL-10, IL-12, TNF-α) and intracellular proteins (Myd88, NF-κß) by Flow Cytometry. Secreted IL-10 was measured by ELISA. CONCLUSION: Our findings confirm not only that the host genetic basis plays a role in the development of a Th2/Th1/Treg response, but also it states A. simplex larval antigens present specific mechanisms to modify the innate response of the host. As allergies share common pathways with the immune response against this particular helminth, our results provide a better understanding into the specific mechanisms of A. simplex allergy related diseases.


Subject(s)
Anisakis/immunology , Antigens/immunology , Immunomodulation , Larva/immunology , Toll-Like Receptors/immunology , Animals , Female , H-2 Antigens , Interleukin-10/metabolism , Interleukin-12/metabolism , Mice, Inbred BALB C , Mice, Inbred C57BL , Toll-Like Receptor 2 , Toll-Like Receptors/agonists
12.
Int J Mol Sci ; 22(15)2021 Jul 23.
Article in English | MEDLINE | ID: mdl-34360627

ABSTRACT

Fucosylation is involved in a wide range of biological processes from cellular adhesion to immune regulation. Although the upregulation of fucosylated glycans was reported in diseased corneas, its implication in ocular surface disorders remains largely unknown. In this study, we analyzed the expression of a fucosylated glycan on the ocular surface in two mouse models of dry eye disease (DED), the NOD.B10.H2b mouse model and the environmental desiccating stress model. We furthermore investigated the effects of aberrant fucosylation inhibition on the ocular surface and DED. Results demonstrated that the level of type 2 H antigen, an α(1,2)-fucosylated glycan, was highly increased in the cornea and conjunctiva both in NOD.B10.H2b mice and in BALB/c mice subjected to desiccating stress. Inhibition of α(1,2)-fucosylation by 2-deoxy-D-galactose (2-D-gal) reduced corneal epithelial defects and increased tear production in both DED models. Moreover, 2-D-gal treatment suppressed the levels of inflammatory cytokines in the ocular surface and the percentages of IFN-γ+CD4+ cells in draining lymph nodes, whereas it did not affect the number of conjunctival goblet cells, the MUC5AC level or the meibomian gland area. Together, the findings indicate that aberrant fucosylation underlies the pathogenesis of DED and may be a novel target for DED therapy.


Subject(s)
Conjunctiva/metabolism , Cornea/metabolism , Dry Eye Syndromes/etiology , Galactose/analogs & derivatives , H-2 Antigens/metabolism , Animals , Conjunctiva/drug effects , Cornea/drug effects , Disease Models, Animal , Drug Evaluation, Preclinical , Dry Eye Syndromes/drug therapy , Dry Eye Syndromes/metabolism , Fucose/metabolism , Galactose/pharmacology , Galactose/therapeutic use , Male , Mice , Mice, Inbred BALB C , Polysaccharides/metabolism
13.
Eur J Immunol ; 51(10): 2531-2534, 2021 10.
Article in English | MEDLINE | ID: mdl-34453339

ABSTRACT

Simultaneous triggering of NK1.1 and MHC class I on NK cells gives a higher Ca2+ flux response compared to triggering the NK1.1 receptor alone. The data suggest a novel costimulatory role for MHC class I molecules on NK cell responses.


Subject(s)
Antigens, Ly/immunology , Calcium/metabolism , Histocompatibility Antigens Class I/immunology , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , NK Cell Lectin-Like Receptor Subfamily B/immunology , Signal Transduction , Animals , H-2 Antigens/genetics , H-2 Antigens/immunology , Mice
14.
Front Immunol ; 12: 630307, 2021.
Article in English | MEDLINE | ID: mdl-33912160

ABSTRACT

The study was aimed at developing an accessible laboratory animal model to elucidate protective and pathological roles of immune mediators during Peste des petits ruminants virus (PPRV) infection. It is because of the critical roles of type I IFNs in anti-viral defense, we assessed the susceptibility of IFN receptor knock out (IFNR KO) mice to PPRV infection. IFNR KO mice were exceedingly susceptible to the infection but WT animals efficiently controlled PPRV. Accordingly, the PPRV infected IFNR KO mice gradually reduced their body weights and succumbed to the infection within 10 days irrespective of the dose and route of infection. The lower infecting doses predominantly induced immunopathological lesions. The viral antigens as well as the replicating PPRV were abundantly present in most of the critical organs such as brain, lungs, heart and kidneys of IFNR KO mice infected with high dose of the virus. Neutrophils and macrophages transported the replicating virus to central nervous system (CNS) and contributed to pathology while the elevated NK and T cell responses directly correlated with the resolution of PPRV infection in WT animals. Using an array of fluorescently labeled H-2Kb tetramers, we discovered four immunogenic epitopes of PPRV. The PPRV-peptides interacted well with H-2Kb in acellular and cellular assay as well as expanded the virus-specific CD8+ T cells in immunized or infected mice. Adoptively transferred CD8+ T cells helped control PPRV in infected mice. Our study therefore established and employed a mouse model for investigating the pathogenesis of PPRV. The model could be useful for elucidating the contribution of immune cells in disease progression as well as to test anti-viral agents.


Subject(s)
Peste-des-Petits-Ruminants/immunology , Animals , Brain/virology , CD8-Positive T-Lymphocytes/immunology , Disease Models, Animal , H-2 Antigens/immunology , Immunity, Innate , Immunization , Lung/virology , Mice , Mice, Inbred C57BL , Peste-des-Petits-Ruminants/mortality , Peste-des-Petits-Ruminants/pathology , Peste-des-petits-ruminants virus/immunology , Receptors, Interferon/physiology , Viral Vaccines/immunology
15.
Hepatology ; 73(2): 759-775, 2021 02.
Article in English | MEDLINE | ID: mdl-32342533

ABSTRACT

BACKGROUND AND AIMS: Growth hormone (GH) is important for liver regeneration after partial hepatectomy (PHx). We investigated this process in C57BL/6 mice that express different forms of the GH receptor (GHR) with deletions in key signaling domains. APPROACH AND RESULTS: PHx was performed on C57BL/6 mice lacking GHR (Ghr-/- ), disabled for all GH-dependent Janus kinase 2 signaling (Box1-/- ), or lacking only GH-dependent signal transducer and activator of transcription 5 (STAT5) signaling (Ghr391-/- ), and wild-type littermates. C57BL/6 Ghr-/- mice showed striking mortality within 48 hours after PHx, whereas Box1-/- or Ghr391-/- mice survived with normal liver regeneration. Ghr-/- mortality was associated with increased apoptosis and elevated natural killer/natural killer T cell and macrophage cell markers. We identified H2-Bl, a key immunotolerance protein, which is up-regulated by PHx through a GH-mediated, Janus kinase 2-independent, SRC family kinase-dependent pathway. GH treatment was confirmed to up-regulate expression of the human homolog of H2-Bl (human leukocyte antigen G [HLA-G]) in primary human hepatocytes and in the serum of GH-deficient patients. We find that injury-associated innate immune attack by natural killer/natural killer T cell and macrophage cells are instrumental in the failure of liver regeneration, and this can be overcome in Ghr-/- mice by adenoviral delivery of H2-Bl or by infusion of HLA-G protein. Further, H2-Bl knockdown in wild-type C57BL/6 mice showed elevated markers of inflammation after PHx, whereas Ghr-/- backcrossed on a strain with high endogenous H2-Bl expression showed a high rate of survival following PHx. CONCLUSIONS: GH induction of H2-Bl expression is crucial for reducing innate immune-mediated apoptosis and promoting survival after PHx in C57BL/6 mice. Treatment with HLA-G may lead to improved clinical outcomes following liver surgery or transplantation.


Subject(s)
Growth Hormone/deficiency , H-2 Antigens/metabolism , HLA-G Antigens/metabolism , Liver Regeneration/immunology , Liver/physiology , Animals , Apoptosis/immunology , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cells, Cultured , Coculture Techniques , Gene Knockdown Techniques , H-2 Antigens/genetics , HLA-G Antigens/genetics , HLA-G Antigens/isolation & purification , Hepatectomy , Hepatocytes , Humans , Immunity, Innate , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Liver/surgery , Macrophages/immunology , Macrophages/metabolism , Mice , Natural Killer T-Cells/immunology , Natural Killer T-Cells/metabolism , Primary Cell Culture , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Signal Transduction/genetics , Signal Transduction/immunology
16.
J Virol ; 94(24)2020 11 23.
Article in English | MEDLINE | ID: mdl-32999036

ABSTRACT

Intracranial (i.c.) infection of susceptible C57BL/6 mice with the neurotropic JHM strain of mouse hepatitis virus (JHMV) (a member of the Coronaviridae family) results in acute encephalomyelitis and viral persistence associated with an immune-mediated demyelinating disease. The present study was undertaken to better understand the molecular pathways evoked during innate and adaptive immune responses as well as the chronic demyelinating stage of disease in response to JHMV infection of the central nervous system (CNS). Using single-cell RNA sequencing analysis (scRNAseq) on flow-sorted CD45-positive (CD45+) cells enriched from brains and spinal cords of experimental mice, we demonstrate the heterogeneity of the immune response as determined by the presence of unique molecular signatures and pathways involved in effective antiviral host defense. Furthermore, we identify potential genes involved in contributing to demyelination as well as remyelination being expressed by both microglia and macrophages. Collectively, these findings emphasize the diversity of the immune responses and molecular networks at defined stages following viral infection of the CNS.IMPORTANCE Understanding the immunological mechanisms contributing to both host defense and disease following viral infection of the CNS is of critical importance given the increasing number of viruses that are capable of infecting and replicating within the nervous system. With this in mind, the present study was undertaken to evaluate the molecular signatures of immune cells within the CNS at defined times following infection with a neuroadapted murine coronavirus using scRNAseq. This approach has revealed that the immunological landscape is diverse, with numerous immune cell subsets expressing distinct mRNA expression profiles that are, in part, dictated by the stage of infection. In addition, these findings reveal new insight into cellular pathways contributing to control of viral replication as well as to neurologic disease.


Subject(s)
Central Nervous System Infections/immunology , Central Nervous System Infections/virology , Coronavirus Infections/immunology , Coronavirus Infections/virology , Host-Pathogen Interactions/immunology , Murine hepatitis virus/physiology , Animals , Central Nervous System Infections/genetics , Central Nervous System Infections/pathology , Computational Biology/methods , Coronavirus Infections/genetics , Coronavirus Infections/pathology , Encephalomyelitis/genetics , Encephalomyelitis/immunology , Encephalomyelitis/pathology , Encephalomyelitis/virology , Gene Expression Profiling , H-2 Antigens/genetics , H-2 Antigens/immunology , Host-Pathogen Interactions/genetics , Immunity, Innate , Mice , Sequence Analysis, RNA , Single-Cell Analysis
17.
Nat Immunol ; 21(11): 1384-1396, 2020 11.
Article in English | MEDLINE | ID: mdl-32989327

ABSTRACT

T follicular helper (TFH) cells are critical in adaptive immune responses to pathogens and vaccines; however, what drives the initiation of their developmental program remains unclear. Studies suggest that a T cell antigen receptor (TCR)-dependent mechanism may be responsible for the earliest TFH cell-fate decision, but a critical aspect of the TCR has been overlooked: tonic TCR signaling. We hypothesized that tonic signaling influences early TFH cell development. Here, two murine TCR-transgenic CD4+ T cells, LLO56 and LLO118, which recognize the same antigenic peptide presented on major histocompatibility complex molecules but experience disparate strengths of tonic signaling, revealed low tonic signaling promotes TFH cell differentiation. Polyclonal T cells paralleled these findings, with naive Nur77 expression distinguishing TFH cell potential. Two mouse lines were also generated to both increase and decrease tonic signaling strength, directly establishing an inverse relationship between tonic signaling strength and TFH cell development. Our findings elucidate a central role for tonic TCR signaling in early TFH cell-lineage decisions.


Subject(s)
Receptors, Antigen, T-Cell/metabolism , Signal Transduction , T Follicular Helper Cells/immunology , T Follicular Helper Cells/metabolism , Animals , Antigens/immunology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Cell Differentiation/immunology , H-2 Antigens/immunology , Immunization , Immunophenotyping , Lymphocyte Activation/immunology , Mice , Mice, Knockout , Mice, Transgenic , Peptides/immunology
18.
J Immunol Res ; 2020: 9686143, 2020.
Article in English | MEDLINE | ID: mdl-32953894

ABSTRACT

BACKGROUND: Nonspecific immunosuppressive therapy for graft rejection and graft-versus-host disease (GVHD) is often accompanied by severe side effects such as opportunistic infections and cancers. Several approaches have been developed to suppress transplantation reactions using tolerogenic cells, including induction of FoxP3+ Tregs with antigen-loaded dendritic cells (DCs) and induction of CD4+IL-10+ cells with interleukin IL-10-producing DCs. Here, we assessed the effectiveness of both approaches in the suppression of graft rejection and GVHD. METHODS: IL-10-producing DCs were generated by the transfection of DCs with DNA constructs encoding mouse IL-10. Antigen-loaded DCs from C57BL/6 mice were generated by transfection with DNA constructs encoding antigenic determinants from the H2 locus of CBA mice which differ from the homologous antigenic determinants of C57BL/6 mice. RESULTS: We found that both IL-10-producing DCs and antigen-loaded immature DCs could suppress graft rejection and GVHD but through distinct nonspecific and antigen-specific mechanisms, respectively. Discussion. We provide data that the novel approach for DCs antigen loading using DNA constructs encoding distinct homologous determinants derived from major histocompatibility complex genes is effective in antigen-specific suppression of transplantation reactions. Such an approach eliminates the necessity of donor material use and may be useful in immunosuppressive therapy side effects prevention.


Subject(s)
Dendritic Cells/immunology , Epitopes/immunology , H-2 Antigens/immunology , Immune Tolerance , Animals , Dendritic Cells/metabolism , Disease Models, Animal , Epitopes/genetics , Female , Gene Order , Graft Rejection/immunology , Graft vs Host Disease/diagnosis , Graft vs Host Disease/etiology , Graft vs Host Disease/therapy , H-2 Antigens/genetics , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Plasmids/genetics , T-Lymphocyte Subsets , Transfection , Transplantation, Homologous
19.
Science ; 369(6506): 936-942, 2020 08 21.
Article in English | MEDLINE | ID: mdl-32820119

ABSTRACT

Intestinal microbiota have been proposed to induce commensal-specific memory T cells that cross-react with tumor-associated antigens. We identified major histocompatibility complex (MHC) class I-binding epitopes in the tail length tape measure protein (TMP) of a prophage found in the genome of the bacteriophage Enterococcus hirae Mice bearing E. hirae harboring this prophage mounted a TMP-specific H-2Kb-restricted CD8+ T lymphocyte response upon immunotherapy with cyclophosphamide or anti-PD-1 antibodies. Administration of bacterial strains engineered to express the TMP epitope improved immunotherapy in mice. In renal and lung cancer patients, the presence of the enterococcal prophage in stools and expression of a TMP-cross-reactive antigen by tumors correlated with long-term benefit of PD-1 blockade therapy. In melanoma patients, T cell clones recognizing naturally processed cancer antigens that are cross-reactive with microbial peptides were detected.


Subject(s)
Antigens, Neoplasm/immunology , Bacteriophages/immunology , Enterococcus hirae/virology , Gastrointestinal Microbiome/immunology , Histocompatibility Antigens Class I/immunology , Immunotherapy/methods , Neoplasms/therapy , Viral Tail Proteins/immunology , Animals , Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents, Alkylating/therapeutic use , CD8-Positive T-Lymphocytes/immunology , Cross Reactions , Cyclophosphamide/therapeutic use , Epitopes/immunology , Feces/virology , H-2 Antigens/immunology , Humans , Mice , Neoplasms/diet therapy , Neoplasms/immunology , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/immunology , Viral Tail Proteins/therapeutic use
20.
J Immunol ; 205(5): 1228-1238, 2020 09 01.
Article in English | MEDLINE | ID: mdl-32737149

ABSTRACT

Theiler's murine encephalomyelitis virus (TMEV) infection of the CNS is cleared in C57BL/6 mice by a CD8 T cell response restricted by the MHC class I molecule H-2Db The identity and function of the APC(s) involved in the priming of this T cell response is (are) poorly defined. To address this gap in knowledge, we developed an H-2Db LoxP-transgenic mouse system using otherwise MHC class I-deficient C57BL/6 mice, thereby conditionally ablating MHC class I-restricted Ag presentation in targeted APC subpopulations. We observed that CD11c+ APCs are critical for early priming of CD8 T cells against the immunodominant TMEV peptide VP2121-130 Loss of H-2Db on CD11c+ APCs mitigates the CD8 T cell response, preventing early viral clearance and immunopathology associated with CD8 T cell activity in the CNS. In contrast, animals with H-2Db-deficient LysM+ APCs retained early priming of Db:VP2121-130 epitope-specific CD8 T cells, although a modest reduction in immune cell entry into the CNS was observed. This work establishes a model enabling the critical dissection of H-2Db-restricted Ag presentation to CD8 T cells, revealing cell-specific and temporal features involved in the generation of CD8 T cell responses. Employing this novel system, we establish CD11c+ cells as pivotal to the establishment of acute antiviral CD8 T cell responses against the TMEV immunodominant epitope VP2121-130, with functional implications both for T cell-mediated viral control and immunopathology.


Subject(s)
Antigens, Viral/immunology , CD8-Positive T-Lymphocytes/immunology , Cardiovirus Infections/immunology , Genes, MHC Class I/immunology , H-2 Antigens/immunology , Theilovirus/immunology , Animals , Antigen Presentation , Capsid Proteins/immunology , Epitopes, T-Lymphocyte/immunology , Immunodominant Epitopes/immunology , Kinetics , Mice , Mice, Inbred C57BL , Mice, Transgenic
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