ABSTRACT
Mixed lymphocyte culture under the blockade of CD80/CD86-CD28 co-stimulation induces anergic (completely hyporesponsive) T cells with immune suppressive function (inducible suppressing T cells: iTS cells). Previously, iTS cell therapy has demonstrated outstanding benefits in clinical trials for organ transplantation. Here, we examined whether peptide antigen-specific iTS cells are inducible. DO 11.10 iTS cells were obtained from splenocytes of BALB/c DO 11.10 mice by stimulation with OVA peptide and antagonistic anti-CD80/CD86 mAbs. When DO 11.10 iTS or Foxp3- DO 11.10 iTS cells were stimulated with OVA, these cells produced IL-13, but not IL-4. DO 11.10 iTS cells decreased IL-4 and increased IL-13 production from OVA-stimulated naïve DO 11.10 splenocytes. When Foxp3+ DO 11.10 iTS cells were prepared, these cells significantly inhibited the production of IL-4 and IL-13 compared with freshly isolated Foxp3+ DO 11.10 T cells. Moreover, an increase in the population expressing OX40, ICOS, and 4-1BB suggested activation of Foxp3+ DO 11.10 iTS cells. Thus, blockade of CD80/CD86-CD28 co-stimulation during peptide antigen stimulation augments the inhibitory function of Foxp3+ regulatory T cells, and does not induce anergic Foxp3- conventional T cells. Peptide-specific Foxp3+ regulatory iTS cells could be useful for the treatment of allergic and autoimmune diseases without adverse effects.
Subject(s)
B7-1 Antigen , B7-2 Antigen , CD28 Antigens , T-Lymphocytes, Regulatory , Animals , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , CD28 Antigens/immunology , CD28 Antigens/metabolism , Mice , B7-1 Antigen/metabolism , B7-1 Antigen/immunology , B7-2 Antigen/metabolism , B7-2 Antigen/immunology , Mice, Inbred BALB C , Forkhead Transcription Factors/metabolism , Peptides/pharmacology , Peptides/immunology , Lymphocyte Activation/immunology , Interleukin-4/metabolism , Interleukin-4/immunology , Interleukin-13/metabolism , Interleukin-13/immunology , Ovalbumin/immunology , Spleen/immunology , Spleen/cytology , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/immunologyABSTRACT
Obesity is a leading risk factor for progression and metastasis of many cancers1,2, yet can in some cases enhance survival3-5 and responses to immune checkpoint blockade therapies, including anti-PD-1, which targets PD-1 (encoded by PDCD1), an inhibitory receptor expressed on immune cells6-8. Although obesity promotes chronic inflammation, the role of the immune system in the obesity-cancer connection and immunotherapy remains unclear. It has been shown that in addition to T cells, macrophages can express PD-19-12. Here we found that obesity selectively induced PD-1 expression on tumour-associated macrophages (TAMs). Type I inflammatory cytokines and molecules linked to obesity, including interferon-γ, tumour necrosis factor, leptin, insulin and palmitate, induced macrophage PD-1 expression in an mTORC1- and glycolysis-dependent manner. PD-1 then provided negative feedback to TAMs that suppressed glycolysis, phagocytosis and T cell stimulatory potential. Conversely, PD-1 blockade increased the level of macrophage glycolysis, which was essential for PD-1 inhibition to augment TAM expression of CD86 and major histocompatibility complex I and II molecules and ability to activate T cells. Myeloid-specific PD-1 deficiency slowed tumour growth, enhanced TAM glycolysis and antigen-presentation capability, and led to increased CD8+ T cell activity with a reduced level of markers of exhaustion. These findings show that obesity-associated metabolic signalling and inflammatory cues cause TAMs to induce PD-1 expression, which then drives a TAM-specific feedback mechanism that impairs tumour immune surveillance. This may contribute to increased cancer risk yet improved response to PD-1 immunotherapy in obesity.
Subject(s)
Neoplasms , Obesity , Programmed Cell Death 1 Receptor , Tumor-Associated Macrophages , Animals , Female , Humans , Male , Mice , Antigen Presentation/drug effects , B7-2 Antigen/antagonists & inhibitors , B7-2 Antigen/immunology , B7-2 Antigen/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Glycolysis/drug effects , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class II/immunology , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic use , Inflammation Mediators/immunology , Inflammation Mediators/metabolism , Lymphocyte Activation , Mechanistic Target of Rapamycin Complex 1/metabolism , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Mice, Inbred C57BL , Neoplasms/drug therapy , Neoplasms/immunology , Neoplasms/metabolism , Neoplasms/pathology , Obesity/immunology , Obesity/metabolism , Phagocytosis/drug effects , Programmed Cell Death 1 Receptor/metabolism , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Tumor-Associated Macrophages/immunology , Tumor-Associated Macrophages/metabolism , Tumor-Associated Macrophages/drug effectsABSTRACT
When developing a program of preclinical studies of human cell-based drugs intended for adoptive immunotherapy of cancer patients, the biological effect should be substantiated by data describing their immunological action. Administration and study of human autologous dendritic cell vaccine to immunocompetent animals are not adequate in terms of immunological compatibility. It is possible to use immunocompromised, knockout, or transgenic animals or to obtain a homologous cellular product, namely, a preparation based on animal cells using a technology similar to obtaining the original preparation for clinical practice in humans. Within the framework of this study, we have developed a protocol for obtaining a homologous cell product based on animal dendritic cells (mice, rats) according to a similar technology for obtaining human vaccine dendritic cells, and demonstrated the comparability of morphological characteristics and expression of differentiation antigens of dendritic cells (CD11c, CD80, CD86, and CD83) of animals (mice) and humans.
Subject(s)
Cancer Vaccines , Dendritic Cells , Immunotherapy, Adoptive , Animals , Dendritic Cells/immunology , Dendritic Cells/drug effects , Cancer Vaccines/immunology , Mice , Humans , Rats , Immunotherapy, Adoptive/methods , B7-1 Antigen/immunology , B7-1 Antigen/metabolism , B7-1 Antigen/genetics , CD11c Antigen/metabolism , CD11c Antigen/immunology , B7-2 Antigen/metabolism , B7-2 Antigen/immunology , B7-2 Antigen/geneticsABSTRACT
To explore the effect of K33 only mutant ubiquitin (K33O) on bone marrow-derived dendritic cells' (BMDCs') maturity, antigen uptake capability, surface molecule expressions and BMDC-mediated CTL priming, and further investigate the role of PI3K-Akt engaged in K33O-increased BMDC maturation, antigen uptake and presentation, surface molecule expressions and BMDC-based CTL priming. BMDCs were conferred K33O and other ubiquitin mutants (K33R, K48R, K63R-mutant ubiquitin) incubation or LY294002 and wortmannin pretreatment. PI3K-Akt phosphorylation, antigen uptake, antigenic presentation and CD86/MHC class I expression in BMDC were determined by western blot or flow cytometry. BMDC-based CTL proliferation and priming were determined by in vitro mixed lymphocyte reaction (MLR), ex vivo enzyme-linked immunospot assay (Elispot) and flow cytometry with intracellular staining, respectively. The treatment with K33O effectively augmented PI3K-Akt phosphorylation, BMDCs' antigen uptake, antigenic presentation, CD86/MHC class I and CD11c expressions. MLR, Elispot and flow cytometry revealed that K33O treatment obviously enhanced CTL proliferation, CTL priming and perforin/granzyme B expression. The pretreatment with PI3K-Akt inhibitors efficiently abrogated K33O's effects on BMDC. The replenishment of K33 only mutant ubiquitin augments BMDC-mediated CTL priming in bone marrow-derived dendritic cells via PI3K-Akt signalling.
Subject(s)
Antigen Presentation , Bone Marrow Cells , Dendritic Cells , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Signal Transduction , T-Lymphocytes, Cytotoxic , Ubiquitin , Dendritic Cells/immunology , Dendritic Cells/metabolism , Animals , Mice , Proto-Oncogene Proteins c-akt/metabolism , Ubiquitin/metabolism , T-Lymphocytes, Cytotoxic/immunology , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Antigen Presentation/immunology , Mice, Inbred C57BL , Phosphorylation , Lymphocyte Activation , Cell Differentiation , Mutation , Morpholines/pharmacology , Lymphocyte Culture Test, Mixed , Cell Proliferation , B7-2 Antigen/metabolism , B7-2 Antigen/genetics , B7-2 Antigen/immunology , Cells, Cultured , Chromones/pharmacology , Wortmannin/pharmacology , Androstadienes/pharmacologyABSTRACT
The objective of this study is to detect the number of different subsets of TFH and B cells in renal transplant recipients (RTR) with antibody-mediated acute rejection (AMR), acute rejection (AR), chronic rejection (CR), or transplant stable (TS). The present study was a prospective study. The numbers of ICOS +, PD-1+ and IL-21+ TFH, CD86+, CD38+, CD27+, and IgD- B cells in 21 patients with end-stage renal disease (ESRD) and post-transplant times were measured by flow cytometry. The level of serum IL-21 was detected by ELISA. The numbers of circulating CD4+CXCR5+, CD4+CXCR5+ICOS+, CD4+CXCR5+PD-1+, CD4+CXCR5+IL-21+ TFH, CD19+CD86+, and CD19 +CD86+CD38+ B cells as well as the level of serum IL-21 in the AMR, AR, and CR groups at post-transplantation were significantly higher than those at pre-transplantation. In contrast, the number of circulating CD19+CD27+IgD B cells was significantly increased in the TS groups in respect to the other groups. Moreover, the numbers of circulating CD4+CXCR5+IL-21+ TFH cells, CD19+CD86+CD38+ B cells as well as the level of serum IL-21 were positive related to the level of serum Cr while showing negative correlated with the values of eGFR in the AMR groups at post-transplantation for 4 and 12 weeks. Circulating TFH cells may be a biomarker in RTR with AMR, which can promote the differentiation of B cells into plasma cells by activating B cells, thereby promoting disease progression.
Subject(s)
B-Lymphocytes/immunology , Graft Rejection/blood , Kidney Transplantation , T Follicular Helper Cells/immunology , ADP-ribosyl Cyclase 1/immunology , Acute Disease , Adult , B7-2 Antigen/immunology , Biomarkers/blood , Female , Graft Rejection/immunology , Humans , Immunosuppression Therapy , Interleukins/immunology , Kidney Failure, Chronic/therapy , Male , Middle Aged , Transplant RecipientsABSTRACT
In Japan, there is a long history of consumption of miso, a fermented soybean paste, which possesses beneficial effects on human health. However, the mechanism behind these effects is not fully understood. To clarify the effects of miso on immune cells, we evaluated its immunomodulatory activity in mice. Miso did not alter the percentage of B and T cells in the spleen; however, it increased CD69+ B cells, germinal center B cells and regulatory T cells. Anti-DNA immunoglobulin M antibodies, which prevent autoimmune disease, were increased following ingestion of miso. Transcriptome analysis of mouse spleen cells cultured with miso and its raw material revealed that the expression of genes, including interleukin (IL)-10, IL-22 and CD86, was upregulated. Furthermore, intravital imaging of the small intestinal epithelium using a calcium biosensor mouse line indicated that miso induced Ca2+ signaling in a manner similar to that of probiotics. Thus, ingestion of miso strengthened the immune response and tolerance in mice. These results appear to account, at least in part, to the salubrious effects of miso.
Subject(s)
B-Lymphocytes/immunology , Soy Foods , T-Lymphocytes/immunology , Animals , Antigens, CD/immunology , Antigens, Differentiation, T-Lymphocyte/immunology , B7-2 Antigen/immunology , Interleukin-10/immunology , Interleukins/immunology , Lectins, C-Type/immunology , Mice , Interleukin-22ABSTRACT
Numerous studies reported a small subpopulation of TCRαß+CD4-CD8- (double-negative) T cells that exert regulatory functions in the peripheral lymphocyte population. However, the origin of these double-negative T (DNT) cells is controversial. Some researchers reported that DNT cells originated from the thymus, and others argued that these cells are derived from peripheral immune induction. We report a possible mechanism for the induction of nonregulatory CD4+ T cells to become regulatory double-negative T (iDNT) cells in vitro. We found that immature bone marrow dendritic cells (CD86+MHC-II- DCs), rather than mature DCs (CD86+MHC-II+), induced high levels of iDNT cells. The addition of an anti-MHC-II antibody to the CD86+MHC-II+ DC group significantly increased induction. These iDNT cells promoted B cell apoptosis and inhibited B cell proliferation and plasma cell formation. A subgroup of iDNT cells expressed NKG2D. Compared to NKG2D- iDNT cells, NKG2D+ iDNT cells released more granzyme B to enhance B cell regulation. This enhancement may function via NKG2D ligands expressed on B cells following lipopolysaccharide stimulation. These results demonstrate that MHC-II impedes induction, and iDNT cells may be MHC independent. NKG2D expression on iDNT cells enhanced the regulatory function of these cells. Our findings elucidate one possible mechanism of the induction of peripheral immune tolerance and provide a potential treatment for chronic allograft rejection in the future.
Subject(s)
B-Lymphocytes/immunology , CD4 Antigens/immunology , CD8 Antigens/immunology , NK Cell Lectin-Like Receptor Subfamily K/immunology , T-Lymphocytes/immunology , Animals , B7-2 Antigen/genetics , B7-2 Antigen/immunology , B7-2 Antigen/metabolism , CD4 Antigens/genetics , CD4 Antigens/metabolism , CD8 Antigens/genetics , CD8 Antigens/metabolism , Cells, Cultured , Cytokines/genetics , Cytokines/immunology , Cytokines/metabolism , Dendritic Cells/immunology , Dendritic Cells/metabolism , Gene Expression/immunology , Granzymes/genetics , Granzymes/immunology , Granzymes/metabolism , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/immunology , Histocompatibility Antigens Class II/metabolism , Male , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Models, Immunological , NK Cell Lectin-Like Receptor Subfamily K/genetics , NK Cell Lectin-Like Receptor Subfamily K/metabolism , T-Lymphocytes/metabolismABSTRACT
Mononuclear phagocytes (MNPs) participate in inflammation and repair after kidney injury, reflecting their complex nature. Dissection into refined functional subunits has been challenging and would benefit understanding of renal pathologies. Flow cytometric approaches are limited to classifications of either different MNP subsets or functional state. We sought to combine these two dimensions in one protocol that considers functional heterogeneity in each MNP subset. We identified five distinct renal MNP subsets based on a previously described strategy. In vitro polarization of bone marrow-derived macrophages (BMDM) into M1- and M2-like cells suggested functional distinction of CD86 + MHCII + CD206- and CD206 + cells. Combination of both distinction methods identified CD86 + MHCII + CD206- and CD206 + cells in all five MNP subsets, revealing their heterologous nature. Our approach revealed that MNP composition and their functional segmentation varied between different mouse models of kidney injury and, moreover, was dynamically regulated in a time-dependent manner. CD206 + cells from three analyzed MNP subsets had a higher ex vivo phagocytic capacity than CD86 + MHCII + CD206- counterparts, indicating functional uniqueness of each subset. In conclusion, our novel flow cytometric approach refines insights into renal MNP heterogeneity and therefore could benefit mechanistic understanding of renal pathology.
Subject(s)
Flow Cytometry/methods , Phagocytes/metabolism , Animals , Antigens, Surface , B7-2 Antigen/immunology , Genes, MHC Class II/immunology , Kidney/injuries , Kidney/pathology , Lectins, C-Type/immunology , Macrophages/classification , Macrophages/metabolism , Male , Mannose Receptor , Mannose-Binding Lectins/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Phagocytes/classification , Receptors, Cell Surface/immunologyABSTRACT
The role of B cells in antitumor immunity and their impact on emerging immunotherapies is increasingly gaining attention. B-cell effector functions include not only secretion of antibodies, but also presentation of antigens to T cells. A physiologic B-cell subset with immunostimulatory properties was described in humans, defined by a high expression of CD86 and downregulation of CD21. We used multicolor flow cytometry and IHC to elucidate abundance and spatial distribution of these antigen-presenting B cells (BAPC) in blood (peripheral blood mononuclear cells, PBMC) and tumor samples of 237 patients with cancer. Antigen-specific T-cell responses to cancer testis antigens were determined using tetramer staining and sorted BAPCs in FluoroSpot assays for selected patients. We found that BAPCs were increased in the tumor microenvironment of 9 of 10 analyzed cancer types with site-specific variation. BAPCs were not increased in renal cell carcinoma, whereas we found a systemic increase with elevated fractions in tumor-infiltrating lymphocytes (TIL) and PBMCs of patients with colorectal cancer and gastroesophageal adenocarcinoma. BAPCs were localized in lymphoid follicles of tertiary lymphoid structures (TLS) and were enriched in tumors with increased numbers of TLSs. BAPCs isolated from tumor-draining lymph nodes of patients with cancer showed increased percentages of tumor antigen-specific B cells and induced responses of autologous T cells in vitro. Our results highlight the relevance of BAPCs as professional antigen-presenting cells in tumor immunity and provide a mechanistic rationale for the observed correlation of B-cell abundance and response to immune checkpoint inhibition.
Subject(s)
Adenocarcinoma/immunology , Antigen-Presenting Cells/immunology , B-Lymphocytes/immunology , B7-2 Antigen/immunology , Colorectal Neoplasms/immunology , Tertiary Lymphoid Structures/immunology , Adenocarcinoma/pathology , Adult , Aged , Aged, 80 and over , Antigens, Neoplasm/immunology , Colorectal Neoplasms/pathology , Female , Humans , Immunotherapy/methods , Lymphocytes, Tumor-Infiltrating/immunology , Male , Middle Aged , Tumor Microenvironment , Young AdultABSTRACT
Type II interferon gamma (IFNγ) is a pleiotropic cytokine capable of modulating the innate and adaptive immune responses which has been widely characterized in several teleost families. In fish, IFNγ stimulates the expression of cytokines and chemokines associated with the pro-inflammatory response and enhances the production of nitrogen and oxygen reactive species in phagocytic cells. This work studied the effect of IFNγ on the expression of cell-surface markers on splenocytes of Atlantic salmon (Salmo salar). In vitro results showed that subpopulations of mononuclear splenocytes cultured for 15 days were capable of increasing gene expression and protein availability of cell-surface markers such as CD80/86, CD83 and MHC II, after being stimulated with recombinant IFNγ. These results were observed for subpopulations with characteristics associated with monocytes (51%), and features that could be related to lymphocytes (46.3%). In addition, a decrease in the expression of zbtb46 was detected in IFNγ-stimulated splenocytes. Finally, the expression of IFNγ and cell-surface markers was assessed in Atlantic salmon under field conditions. In vivo results showed that the expression of ifnγ increased simultaneously with the up-regulation of cd80/86, cd83 and mhcii during a natural outbreak of Piscirickettsia salmonis. Overall, the results obtained in this study allow us to propose IFNγ as a candidate molecule to stimulate the phenotypic progression of a small population of immune cells, which will increase antigen presenting cells markers. Thereby, modulatory strategies using IFNγ may generate a robust and coordinated immune response in fish against pathogens that affect aquaculture.
Subject(s)
Antigens, CD/metabolism , B7-1 Antigen/metabolism , B7-2 Antigen/metabolism , Histocompatibility Antigens Class II/metabolism , Immunoglobulins/metabolism , Interferon-gamma/immunology , Membrane Glycoproteins/metabolism , Salmo salar/immunology , Spleen/immunology , Animals , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Antigens, CD/genetics , Antigens, CD/immunology , B7-1 Antigen/genetics , B7-1 Antigen/immunology , B7-2 Antigen/genetics , B7-2 Antigen/immunology , Biomarkers/metabolism , Fish Diseases/immunology , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/immunology , Immunoglobulins/genetics , Immunoglobulins/immunology , Interferon-gamma/pharmacology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , Piscirickettsia , Piscirickettsiaceae Infections/immunology , Piscirickettsiaceae Infections/veterinary , Transcription Factors/genetics , Transcription Factors/immunology , Transcription Factors/metabolism , CD83 AntigenABSTRACT
OBJECTIVE: The aim of this study was to examine the effect of gingival mesenchymal stem cells derived exosomes (GMSC-Exos) on lipopolysaccharide/interferon-gamma (LPS/INF-γ)-induced inflammatory macrophages in a high-lipid microenvironment. MATERIALS AND METHODS: Exosomes were obtained by culturing gingival mesenchymal stem cells (GMSCs) in alpha-MEM with exosome-free fetal bovine serum for 48 h. The control group was produced in vitro by inducing human acute monocytic leukemia cells (THP-1 cells) into naïve macrophages (M0). Inflammatory macrophages (M1) were made by activating M0 macrophages with LPS/IFN-γ. These M1 macrophages were treated with oxidized low-density lipoprotein (ox-LDL) to create the high-lipid group, of which some macrophages were further treated with GMSC-Exos for 24 h to form the GMSC-Exos group. Supernatants were collected, and total RNA were extracted for downstream analysis. The expression of surface markers in macrophages were analyzed by flow cytometry. The lipid accumulation level was assessed by oil red O staining. RESULTS: Exosomes were successfully isolated from GMSC medium. The GMSC-Exos group showed lower Tumor Necrosis Factor-α (TNF-α), Interleukin-6 (IL-6), Interleukin-1ß (IL-1ß), and cluster of differentiation 86 (CD86) expression levels than the high-lipid group, and the highest levels of Interleukin-10 (IL-10) among all groups. The GMSC-Exos group showed significant reductions in TNF-α levels than the high-lipid group, and significant escalations in IL-10 levels than the other two groups. Oil red o Staining showed that lipid accumulation in macrophages was inhibited in the GMSC-Exos group. CONCLUSIONS: GMSC-Exos reduce the release level and expression of inflammatory factors, inhibit lipid accumulation, and promote the polarization of pro-inflammatory macrophages into anti-inflammatory phenotype in a high-lipid microenvironment.
Subject(s)
Exosomes , Macrophages/immunology , Mesenchymal Stem Cells , Adolescent , Adult , B7-2 Antigen/immunology , Cell Differentiation , Gingiva/cytology , Humans , Inflammation/immunology , Interleukin-10/immunology , Lipids , Phenotype , THP-1 Cells , Tumor Necrosis Factor-alpha/immunology , Young AdultABSTRACT
CD80 and CD86 are expressed on antigen presenting cells and are required to engage their shared receptor, CD28, for the costimulation of CD4 T cells. It is unclear why two stimulatory ligands with overlapping roles have evolved. CD80 and CD86 also bind the regulatory molecule CTLA-4. We explored the role of CD80 and CD86 in the homeostasis and proliferation of CD4+FoxP3+ regulatory T cells (Treg), which constitutively express high levels of CTLA-4 yet are critically dependent upon CD28 signals. We observed that CD86 was the dominant ligand for Treg proliferation, survival, and maintenance of a regulatory phenotype, with higher expression of CTLA-4, ICOS, and OX40. We also explored whether CD80-CD28 interactions were specifically compromised by CTLA-4 and found that antibody blockade, clinical deficiency of CTLA-4 and CRISPR-Cas9 deletion of CTLA-4 all improved Treg survival following CD80 stimulation. Taken together, our data suggest that CD86 is the dominant costimulatory ligand for Treg homeostasis, despite its lower affinity for CD28, because CD80-CD28 interactions are selectively impaired by the high levels of CTLA-4. These data suggest a cell intrinsic role for CTLA-4 in regulating CD28 costimulation by direct competition for CD80, and indicate that that CD80 and CD86 have discrete roles in CD28 costimulation of CD4 T cells in the presence of high levels of CTLA-4.
Subject(s)
B7-2 Antigen/immunology , CD28 Antigens/immunology , CTLA-4 Antigen/immunology , Homeostasis/immunology , T-Lymphocytes, Regulatory/immunology , B7-2 Antigen/genetics , CD28 Antigens/genetics , CTLA-4 Antigen/genetics , Homeostasis/genetics , Humans , T-Lymphocytes, Regulatory/cytologyABSTRACT
B cell depletion via anti-CD20 antibodies is a highly effective treatment for multiple sclerosis (MS). However, little is known about the maturation/activation stage of the returning B cell population after treatment cessation and the wider effects on other immune cells. In the present study, 15 relapsing-remitting MS patients receiving 1,000 mg of rituximab were included. B, T, and myeloid cells were analyzed before anti-CD20 administration and in different time intervals thereafter over a period of 24 mo. In comparison to the phenotype before anti-CD20 treatment, the reappearing B cell pool revealed a less mature and more activated phenotype: 1) reappearing B cells were significantly enriched in transitional (before: 10.1 ± 1.9%, after: 58.8 ± 5.2%) and mature naive phenotypes (before: 45.5 ± 3.1%, after: 25.1 ± 3.5%); 2) the frequency of memory B cells was reduced (before: 36.7 ± 3.1%, after: 8.9 ± 1.7%); and 3) reappearing B cells showed an enhanced expression of activation markers CD25 (before: 2.1 ± 0.4%, after: 9.3 ± 2.1%) and CD69 (before: 5.9 ± 1.0%, after: 21.4 ± 3.0%), and expressed significantly higher levels of costimulatory CD40 and CD86. T cells showed 1) a persistent increase in naive (CD4+: before: 11.8 ± 1.3%, after: 18.4 ± 3.4%; CD8+: before: 12.5 ± 1.4%, after: 16.5 ± 2.3%) and 2) a decrease in terminally differentiated subsets (CD4+: before: 47.3 ± 3.2%, after: 34.4 ± 3.7%; CD8+: before: 53.7 ± 2.1%, after: 49.1 ± 2.7%).
Subject(s)
Antibodies/administration & dosage , Antigens, CD20/immunology , B-Lymphocytes/immunology , Multiple Sclerosis, Relapsing-Remitting/drug therapy , Multiple Sclerosis, Relapsing-Remitting/immunology , Adult , Antigens, CD/genetics , Antigens, CD/immunology , Antigens, CD20/genetics , Antigens, Differentiation, T-Lymphocyte/genetics , Antigens, Differentiation, T-Lymphocyte/immunology , B-Lymphocytes/cytology , B7-2 Antigen/genetics , B7-2 Antigen/immunology , Female , Humans , Immunologic Memory , Lectins, C-Type/genetics , Lectins, C-Type/immunology , Male , Middle Aged , Multiple Sclerosis, Relapsing-Remitting/genetics , T-Lymphocytes/cytology , T-Lymphocytes/immunologyABSTRACT
AIMS: B cells can promote or inhibit immune responses against breast cancer. We investigated changes in the frequency of B cells with stimulatory or regulatory capacity in breast tumor draining lymph nodes during cancer progression. MAIN METHODS: We isolated mononuclear cells from fresh axillary lymph nodes (LNs) of 44 patients with breast cancer and stained lymphocytes with antibodies against CD19, CD80, CD86, CD39 and CD73. To assess programmed death-1 (PD-1) and PD-ligand 1 (PD-L1) expression, lymphocytes were briefly stimulated, stained for CD19, PD-1 and PD-L1, and examined with flow cytometry. KEY FINDINGS: The frequency of CD80+ B cells was higher in nonmetastatic lymph nodes, while the percentage of CD86+ B cells showed a positive relationship with higher tumor grade and higher numbers of involved LNs. A small proportion of unstimulated B cells expressed PD-1 or PD-L1 but these molecules were rapidly upregulated on B cells following activation. The frequency of stimulated PD-L1+ B cells showed an inverse association with estrogen and progesterone receptor expression and a nonsignificant positive association with tumor grade. In addition, the percentage of unstimulated PD-1+ B cells was higher in patients with higher-grade tumors. CD73 expression on B cells was associated with lower numbers of involved LNs, and the frequency of CD39+ B cells was higher in patients with larger tumors. SIGNIFICANCE: CD86+, CD39+, PD-1+ and PD-L1+ B cells showed associations with poor prognostic factors, therefore their potential role in the suppression of the immune responses against breast cancer should be evaluated in greater detail.
Subject(s)
B-Lymphocyte Subsets/pathology , B-Lymphocytes, Regulatory/pathology , Breast Neoplasms/immunology , Lymph Nodes/pathology , Adult , Aged , Apyrase/immunology , Axilla , B-Lymphocyte Subsets/immunology , B-Lymphocytes, Regulatory/immunology , B7-2 Antigen/immunology , B7-H1 Antigen/metabolism , Breast Neoplasms/diagnosis , Breast Neoplasms/pathology , Female , Flow Cytometry , Humans , Lymph Nodes/cytology , Lymph Nodes/immunology , Middle Aged , Prognosis , Programmed Cell Death 1 Receptor/metabolismABSTRACT
Emerging evidences indicated that the dysbiosis of microbiota was related to the onset of systemic lupus erythematosus (SLE). Bacteroides fragilis (B. fragilis) ATCC 25285, a human commensal, was discovered to improve inflammatory diseases. However, whether B. fragilis (ATCC 25285) has the beneficial effects on the treatment of lupus nephritis has still remained elusive. In the present study, oral treatment with B. fragilis (ATCC 25285) ameliorated the activity of MRL/lpr mice, including decreased levels of autoantibodies and symptoms of lupus nephritis. Furthermore, we demonstrated that treatment with B. fragilis (ATCC 25285) could promote CD1d expression in B cells by Est-1 pathway, while inhibit CD86 expression via SHP-2 signaling pathway to repair the immune response of B cells in MRL/lpr mice. In addition, our findings revealed a possible role of treatment with B. fragilis (ATCC 25285) in relieving intestinal inflammation in MRL/lpr mice. Meanwhile, it was uncovered that B. fragilis (ATCC 25285) restored the Th17/Treg balance in MRL/lpr mice that was consistent with the role of B. fragilis in other autoimmune diseases. Overall, the current study may highlight the potential application of B. fragilis (ATCC 25285) to treat manifestations of SLE in high-risk individuals.
Subject(s)
Antigens, CD1d/metabolism , B-Lymphocytes/microbiology , B7-2 Antigen/metabolism , Bacteroides fragilis/physiology , Lupus Nephritis/therapy , Probiotics , Animals , Antigens, CD1d/immunology , Autoantibodies/blood , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , B7-2 Antigen/immunology , Disease Models, Animal , Female , Lupus Nephritis/immunology , Lupus Nephritis/metabolism , Lupus Nephritis/microbiology , Mice, Inbred C57BL , Mice, Inbred MRL lpr , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Proto-Oncogene Protein c-ets-1/metabolism , Signal Transduction , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , T-Lymphocytes, Regulatory/microbiology , Th17 Cells/immunology , Th17 Cells/metabolism , Th17 Cells/microbiologyABSTRACT
Murine studies have shown the potential for γδ T cells to mediate immunity to Staphylococcus aureus in multiple tissue settings by the secretion of diverse cytokines. However, the role played by γδ T cells in human immune responses to S. aureus is almost entirely unknown. In this study, we establish the capacity of human Vδ2+ γδ T cells for rapid activation in response to S. aureus In coculture with S. aureus-infected monocyte-derived dendritic cells (DCs), Vδ2+ cells derived from peripheral blood rapidly upregulate CD69 and secrete high levels of IFN-γ. DCs mediate this response through direct contact and IL-12 secretion. In turn, IFN-γ released by Vδ2+ cells upregulates IL-12 secretion by DCs in a positive feedback loop. Furthermore, coculture with γδ T cells results in heightened expression of the costimulatory molecule CD86 and the lymph node homing molecule CCR7 on S. aureus-infected DCs. In cocultures of CD4+ T cells with S. aureus-infected DCs, the addition of γδ T cells results in heightened CD4+ T cell activation. Our findings identify γδ T cells as potential key players in the early host response to S. aureus during bloodstream infection, promoting enhanced responses by both innate and adaptive immune cell populations, and support their consideration in the development of host-directed anti-S. aureus treatments.
Subject(s)
Adaptive Immunity/immunology , CD4-Positive T-Lymphocytes/immunology , Lymphocyte Activation/immunology , Receptors, Antigen, T-Cell, gamma-delta/immunology , Staphylococcal Infections/immunology , Staphylococcus aureus/immunology , Antigens, CD/immunology , Antigens, Differentiation, T-Lymphocyte/immunology , B7-2 Antigen/immunology , Cells, Cultured , Dendritic Cells/immunology , Humans , Interferon-gamma/immunology , Interleukin-12/immunology , Lectins, C-Type/immunology , Monocytes/immunology , Receptors, CCR7/immunology , Up-Regulation/immunologyABSTRACT
A contribution of epigenetic modifications to B cell tolerance has been proposed but not directly tested. Here we report that deficiency of ten-eleven translocation (Tet) DNA demethylase family members Tet2 and Tet3 in B cells led to hyperactivation of B and T cells, autoantibody production and lupus-like disease in mice. Mechanistically, in the absence of Tet2 and Tet3, downregulation of CD86, which normally occurs following chronic exposure of self-reactive B cells to self-antigen, did not take place. The importance of dysregulated CD86 expression in Tet2- and Tet3-deficient B cells was further demonstrated by the restriction, albeit not complete, on aberrant T and B cell activation following anti-CD86 blockade. Tet2- and Tet3-deficient B cells had decreased accumulation of histone deacetylase 1 (HDAC1) and HDAC2 at the Cd86 locus. Thus, our findings suggest that Tet2- and Tet3-mediated chromatin modification participates in repression of CD86 on chronically stimulated self-reactive B cells, which contributes, at least in part, to preventing autoimmunity.
Subject(s)
Autoimmunity/immunology , B-Lymphocytes/immunology , B7-2 Antigen/immunology , DNA-Binding Proteins/immunology , Dioxygenases/immunology , Proto-Oncogene Proteins/immunology , Animals , Autoimmune Diseases/immunology , Epigenesis, Genetic/immunology , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Mice, TransgenicABSTRACT
BACKGROUND: Traditionally, the transcriptomic and proteomic characterisation of CD4+ T cells at the single-cell level has been performed by two largely exclusive types of technologies: single-cell RNA sequencing (scRNA-seq) and antibody-based cytometry. Here, we present a multi-omics approach allowing the simultaneous targeted quantification of mRNA and protein expression in single cells and investigate its performance to dissect the heterogeneity of human immune cell populations. METHODS: We have quantified the single-cell expression of 397 genes at the mRNA level and up to 68 proteins using oligo-conjugated antibodies (AbSeq) in 43,656 primary CD4+ T cells isolated from the blood and 31,907 CD45+ cells isolated from the blood and matched duodenal biopsies. We explored the sensitivity of this targeted scRNA-seq approach to dissect the heterogeneity of human immune cell populations and identify trajectories of functional T cell differentiation. RESULTS: We provide a high-resolution map of human primary CD4+ T cells and identify precise trajectories of Th1, Th17 and regulatory T cell (Treg) differentiation in the blood and tissue. The sensitivity provided by this multi-omics approach identified the expression of the B7 molecules CD80 and CD86 on the surface of CD4+ Tregs, and we further demonstrated that B7 expression has the potential to identify recently activated T cells in circulation. Moreover, we identified a rare subset of CCR9+ T cells in the blood with tissue-homing properties and expression of several immune checkpoint molecules, suggestive of a regulatory function. CONCLUSIONS: The transcriptomic and proteomic hybrid technology described in this study provides a cost-effective solution to dissect the heterogeneity of immune cell populations at extremely high resolution. Unexpectedly, CD80 and CD86, normally expressed on antigen-presenting cells, were detected on a subset of activated Tregs, indicating a role for these co-stimulatory molecules in regulating the dynamics of CD4+ T cell responses.
Subject(s)
B7-1 Antigen/immunology , B7-2 Antigen/immunology , T-Lymphocytes, Regulatory/immunology , Adolescent , Adult , Female , Forkhead Transcription Factors/genetics , Humans , Male , Proteome , RNA , RNA-Seq , Single-Cell Analysis , TranscriptomeABSTRACT
BACKGROUND: Cytotoxic T lymphocyte-associated antigen-4-Ig (CTLA-4-Ig) competes with CD28 for binding CD80/CD86 on antigen-presenting cells (APCs) to limit T cell activation. B cells are believed to be important APCs in the pathogenesis of autoimmune diseases and express CD80/CD86 after activation; however, relatively little is known about the effect of CTLA-4-Ig on B cells. This study tested the impact of CTLA-4-Ig on human B cell responses. METHODS: Human blood B cells were purified from healthy donors and activated in the presence of CTLA-4-Ig or the L6-Ig control protein in vitro. RT-q-PCR and immunofluorescence staining were performed to detect activation marker expression. ELISA was conducted to measure cytokine secretion. The CD80/CD86 levels on the surface of the memory B cells in the blood of 18 patients with rheumatoid arthritis (RA) were detected using immunofluorescence staining. RESULTS: CTLA-4-Ig suppressed the expression of Staphylococcus aureus (SAC)-induced CD80, CD86, TNFA, and IL6 in human B cells at the transcriptional level. Furthermore, CTLA-4-Ig concomitantly decreased SAC-induced CD80/CD86 surface expression on and TNF-α and IL-6 secretion from B cells. On the other hand, T cell-dependent (TD) stimulation-induced B cell activation, proliferation, plasma cell differentiation, and antibody secretion were not affected by CTLA-4-Ig. As expected, TD stimulation-induced surface CD80 was hindered by CTLA-4-Ig. Notably, a blockade of CD80/CD86 on the surface of the memory B cells was observed in the patients with RA after abatacept (CTLA-4-Ig) treatment. In a portion of the RA patients, restoration of CD80/CD86 staining on the surface of the memory B was detected starting in the 3rd month of abatacept treatment. Interestingly, the surface levels of CD80/CD86 on the patients' memory B cells positively correlated with disease activity. CONCLUSIONS: We found that CTLA-4-Ig directly suppressed SAC-induced B cell activation in vitro. Obstruction of CD80 and CD86 on the surface of the memory B cells was detected in the RA patients after abatacept treatment. Blocking CD80/CD86 on B cells by CTLA-4-Ig may hinder T cell activation and associated with the disease activity of RA in vivo. Our findings indicate that CTLA-4-Ig may regulate humoral responses by modulating B cell activation and interfering T cell-B cell interaction.
Subject(s)
Abatacept/pharmacology , B-Lymphocytes/drug effects , B7-2 Antigen/metabolism , CD28 Antigens/metabolism , Cytokines/metabolism , T-Lymphocytes, Cytotoxic/drug effects , Abatacept/immunology , Abatacept/metabolism , Adult , Antigen-Presenting Cells/drug effects , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/pathology , B-Lymphocytes/metabolism , B-Lymphocytes/microbiology , B7-2 Antigen/genetics , B7-2 Antigen/immunology , CD28 Antigens/genetics , CD28 Antigens/immunology , Cells, Cultured , Cytokines/genetics , Cytokines/immunology , Female , Gene Expression/drug effects , Gene Expression/immunology , Humans , Immune Checkpoint Inhibitors/immunology , Immune Checkpoint Inhibitors/pharmacology , Male , Middle Aged , Staphylococcus aureus/immunology , Staphylococcus aureus/physiology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolismABSTRACT
BACKGROUND: The use of anti-B cell based therapies in immune-mediated diseases targeting general B cell markers or molecules important for B cell function has increased the clinical needs of monitoring B cell subpopulations. RESULTS: We analyzed the expression profile of cell surface markers CD86 and B and T lymphocyte attenuator (BTLA) in B cell subtypes using flow cytometry, including naïve, transitional, switched memory, non-switched memory and double-negative memory B cells and plasmablasts, and investigated the dependence of age and sex in a healthy adult blood donor population. The switched memory B cell subtype displayed a divergent expression of the markers, with increased CD86 and decreased BTLA as compared to non-switched and double negative memory cells, as well as compared to naïve B cells. Plasmablasts expressed highly increased CD86 compared to all other subtypes and a decreased expression of BTLA compared to naïve cells, but still higher compared to the memory cell populations. Transitional B cells had CD86 and BTLA expression similar to the other naïve cells. CONCLUSIONS: We show divergent expression of CD86 and BTLA in memory cells and plasmablasts compared to naïve B cells independent of age and sex. Furthermore, a similarly divergent difference of expression pattern was seen between the memory cell subtypes, altogether indicating that the combination of CD86 and BTLA might be markers for a permissive activation state. We suggest the combination of CD86 and BTLA expression on B cell subtypes as a potentially important tool in monitoring the status of B cell subtypes before and after treatments influencing the B cell compartment.