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Luo, Z., Thorvaldson, L., Blixt, M. & Singh, K. (2019). Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry. Journal of Visualized Experiments (144), Article ID e58848.
Open this publication in new window or tab >>Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry
2019 (English)In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, no 144, article id e58848Article in journal (Refereed) Published
Abstract [en]

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Place, publisher, year, edition, pages
JOURNAL OF VISUALIZED EXPERIMENTS, 2019
Keywords
Immunology and Infection, Issue 144, Single cell preparation, regulatory T cell, flow cytometry, Foxp3, Helios, Neuropilin 1
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-382478 (URN)10.3791/58848 (DOI)000462905000030 ()30882793 (PubMedID)
Funder
Swedish Research CouncilEXODIAB - Excellence of Diabetes Research in SwedenSwedish Diabetes AssociationSwedish Child Diabetes Foundation
Available from: 2019-04-29 Created: 2019-04-29 Last updated: 2019-04-29Bibliographically approved
He, Q., Li, X., Singh, K., Luo, Z., Meija-Cordova, M., Jamalpour, M., . . . Welsh, M. (2019). The Cdh5-CreERT2 transgene causes conditional Shb gene deletion in hematopoietic cells with consequences for immune cell responses to tumors. Scientific Reports, 9, Article ID 7548.
Open this publication in new window or tab >>The Cdh5-CreERT2 transgene causes conditional Shb gene deletion in hematopoietic cells with consequences for immune cell responses to tumors
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2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 7548Article in journal (Refereed) Published
Abstract [en]

The tamoxifen-responsive conditional Cdh5-CreERT2 is commonly used for endothelial cell specific conditional deletion of loxP-flanked gene sequences. To address the role of endothelial cell Shb gene for B16F10 melanoma immune responses, tamoxifen-injected Cdh5-CreERT2/WT and Cdh5-CreERT2/Shbflox/flox mice received subcutaneous tumor cell injections. We observed a decrease of tumor myeloid cell Shb mRNA in the tamoxifen treated Cdh5-CreERT2/Shbflox/flox mice, which was not present when the mice had undergone a preceding bone marrow transplantation using wild type bone marrow. Differences in CD4+/FoxP3+ Tregs were similarly abolished by a preceding bone marrow transplantation. In ROSA26-mTmG mice, Cdh5-CreERT2 caused detectable floxing in certain bone marrow populations and in spleen cells. Floxing in bone marrow could be detected two months after tamoxifen treatment. In the spleen, however, floxing was undetectable two months after tamoxifen treatment, suggesting that Cdh5-CreERT2 is operating in a non-renewable population of hematopoietic cells in this organ. These data suggest that conditional gene deletion in hematopoietic cells is a potential confounder in experiments attempting to assess the role of endothelial specific effects. A cautious approach to achieve an endothelial-specific phenotype would be to adopt a strategy that includes a preceding bone marrow transplantation.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-383581 (URN)10.1038/s41598-019-44039-z (DOI)000468171100043 ()31101877 (PubMedID)
Funder
Swedish Cancer Society, 180767Swedish Research Council, 2016-01085EU, FP7, Seventh Framework Programme, 312325EXODIAB - Excellence of Diabetes Research in SwedenErnfors Foundation
Available from: 2019-05-17 Created: 2019-05-17 Last updated: 2019-06-24Bibliographically approved
Li, X., Singh, K., Luo, Z., Mejia Cordova, M., Jamalpour, M., Lindahl, B., . . . Welsh, M. (2018). Pro-tumoral immune cell alterations in wild type and Shb-deficient mice in response to 4T1 breast carcinomas. OncoTarget, 9(27), 18720-18733
Open this publication in new window or tab >>Pro-tumoral immune cell alterations in wild type and Shb-deficient mice in response to 4T1 breast carcinomas
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2018 (English)In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 9, no 27, p. 18720-18733Article in journal (Refereed) Published
Abstract [en]

To assess mechanisms responsible for breast carcinoma metastasis, 4T1 breast carcinomas were grown orthotopically in wild type or Shb knockout mice. Tumor growth, metastasis, vascular characteristics and immune cell properties were analyzed. Absence of Shb did not affect tumor growth although it increased lung metastasis. Shb knockout mouse tumors showed decreased redness and less developed vascular plexa located at the periphery of the tumors. No difference in overall tumor vascular density, leakage or pericyte coverage was noted between the genotypes although the average vessel size was smaller in the knockout. Tumors induced an increase of CD11b+ cells in spleen, lymph node, thymus, bone marrow and blood. Numbers of Shb knockout CD11b/CD8+ cells were decreased in lymph nodes and bone marrow of tumor bearing mice. Mice with tumors had reduced numbers of CD4+ lymphocytes in blood/lymphoid organs, whereas in most of these locations the proportion of CD4+ cells co-expressing FoxP3 was increased, suggesting a relative increase in Treg cells. This finding was reinforced by increased blood interleukin-35 (IL-35) in wild type tumor bearing mice. Shb knockout blood showed in addition an increased proportion of IL-35 expressing Treg cells, supporting the notion that absence of Shb further promotes tumor evasion from immune cell recognition. This could explain the increased number of lung metastases observed under these conditions. In conclusion, 4T1 tumors alter immune cell responses that promote tumor expansion, metastasis and escape from T cell recognition in an Shb dependent manner. 

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-347019 (URN)10.18632/oncotarget.24643 (DOI)
Available from: 2018-03-23 Created: 2018-03-23 Last updated: 2018-04-12Bibliographically approved
Espes, D., Singh, K., Sandler, S. & Carlsson, P.-O. (2017). Increased Interleukin-35 Levels in Patients With Type 1 Diabetes With Remaining C-Peptide. Diabetes Care, 40(8), 1090-1095
Open this publication in new window or tab >>Increased Interleukin-35 Levels in Patients With Type 1 Diabetes With Remaining C-Peptide
2017 (English)In: Diabetes Care, ISSN 0149-5992, E-ISSN 1935-5548, Vol. 40, no 8, p. 1090-1095Article in journal (Refereed) Published
Abstract [en]

OBJECTIVE Many patients with long-standing type 1 diabetes have remaining functional β-cells. This study investigated immunological differences between patients with or without measurable remaining endogenous insulin production after ≥10 years duration of disease.

RESEARCH DESIGN AND METHODS Patients (n = 113; ≥18 years of age) with type 1 diabetes and with disease duration of ≥10 years were recruited at Uppsala University Hospital. Residual β-cell function was determined with an ultrasensitive C-peptide ELISA. Circulating cytokines, including interleukin-35 (IL-35), were determined in plasma. Additional blood samples were collected from 14 of the identified C-peptide–positive patients and 12 of the C-peptide–negative patients, as well as from 15 healthy control subjects, and were used for immediate investigation of peripheral blood mononuclear cells.

RESULTS The blood concentration of the cytokine IL-35 was markedly lower in C-peptide–negative patients, and this was associated with a simultaneous decrease in the proportion of IL-35+ regulatory T cells (Tregs), IL-35+ regulatory B cells, and IL-35–producing CD8+Foxp3+ cells. IL-35 has previously been shown to maintain the phenotype of Tregs, block the differentiation of T-helper 17 cells, and thereby dampen immune assaults to β-cells. We found that the proportions of IL-17a+ cells among the Tregs, CD4+ T cells, and CD8+ T cells were lower in the C-peptide–positive patients.

CONCLUSIONS Patients with remaining endogenous β-cell function after >10 years duration of type 1 diabetes differ immunologically from other patients with long-standing type 1 diabetes. In particular, they have a much higher IL-35 production.

National Category
Medical and Health Sciences
Research subject
Immunology
Identifiers
urn:nbn:se:uu:diva-329523 (URN)10.2337/dc16-2121 (DOI)000406014200026 ()28620093 (PubMedID)
Funder
Swedish Research Council, 55X-15043Swedish Research Council, 921-2014-7054EXODIAB - Excellence of Diabetes Research in SwedenSwedish Diabetes AssociationTorsten Söderbergs stiftelseNovo NordiskSwedish Child Diabetes Foundation
Available from: 2017-09-18 Created: 2017-09-18 Last updated: 2017-11-02Bibliographically approved
Luo, Z., Varli, S., Enström, E., Thorvaldson, L., Blixt, M., Hansell, P., . . . Singh, K. (2017). Kinetics of innate immune and regulatory T cells responses in experimental diabetic nephropathy. Paper presented at 44th Annual Meeting of the Scandinavian-Society-for-Immunology (SSI), OCT 17-20, 2017, Stockholm, SWEDEN. Scandinavian Journal of Immunology, 86(4), 304-304
Open this publication in new window or tab >>Kinetics of innate immune and regulatory T cells responses in experimental diabetic nephropathy
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2017 (English)In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 86, no 4, p. 304-304Article in journal, Meeting abstract (Other academic) Published
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-346962 (URN)000411865200135 ()
Conference
44th Annual Meeting of the Scandinavian-Society-for-Immunology (SSI), OCT 17-20, 2017, Stockholm, SWEDEN
Available from: 2018-03-23 Created: 2018-03-23 Last updated: 2018-03-23Bibliographically approved
Mejia Cordova, M., Soläng, C., Luo, Z., Blixt, M., Thorvaldson, L., Sandler, S. & Singh, K. (2017). Kinetics of the innate immune cell responses in experimental Type 1 Diabetes. Paper presented at 44th Annual Meeting of the Scandinavian-Society-for-Immunology (SSI), OCT 17-20, 2017, Stockholm, SWEDEN. Scandinavian Journal of Immunology, 86(4), 303-304
Open this publication in new window or tab >>Kinetics of the innate immune cell responses in experimental Type 1 Diabetes
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2017 (English)In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 86, no 4, p. 303-304Article in journal, Meeting abstract (Other academic) Published
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-346961 (URN)000411865200134 ()
Conference
44th Annual Meeting of the Scandinavian-Society-for-Immunology (SSI), OCT 17-20, 2017, Stockholm, SWEDEN
Available from: 2018-03-23 Created: 2018-03-23 Last updated: 2018-03-23Bibliographically approved
Digre, A., Singh, K., Åbrink, M., Reijmers, R. M., Sandler, S., Vlodavsky, I. & Li, J.-P. (2017). Overexpression of heparanase enhances T lymphocyte activities and intensifies the inflammatory response in a model of murine rheumatoid arthritis. Scientific Reports, 7, Article ID 46229.
Open this publication in new window or tab >>Overexpression of heparanase enhances T lymphocyte activities and intensifies the inflammatory response in a model of murine rheumatoid arthritis
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 46229Article in journal (Refereed) Published
Abstract [en]

Heparanase is an endo-glucuronidase that degrades heparan sulfate chains. The enzyme is expressed at a low level in normal organs; however, elevated expression of heparanase has been detected in several inflammatory conditions, e.g. in the synovial joints of rheumatoid arthritis (RA) patients. Herein, we have applied the model of collagen-induced arthritis (CIA) to transgenic mice overexpressing human heparanase (Hpa-tg) along with wildtype (WT) mice. About 50 % of the induced animals developed clinical symptoms, i.e. swelling of joints, and there were no differences between the Hpa-tg and WT mice in the incidence of disease. However, Hpa-tg mice displayed an earlier response and developed more severe symptoms. Examination of cells from thymus, spleen and lymph nodes revealed increased innate and adaptive immune responses of the Hpa-tg mice, reflected by increased proportions of macrophages, antigen presenting cells and plasmacytoid dendritic cells as well as Helios-positive CD4+ and CD8+ T cells. Furthermore, splenic lymphocytes from Hpa-tg mice showed higher proliferation activity. Our results suggest that elevated expression of heparanase augmented both the innate and adaptive immune system and propagated inflammatory reactions in the murine RA model.

Keywords
Collagen-induced arthritis, Heparanase, Heparan Sulfate, Flow cytometry, T cells
National Category
Immunology in the medical area
Research subject
Immunology; Medical Science
Identifiers
urn:nbn:se:uu:diva-315694 (URN)10.1038/srep46229 (DOI)000398988100001 ()28401953 (PubMedID)
Funder
Swedish Research Council, 2015-02595; K2012-56X-15046-09-4Swedish Research Council, 521-2011-3533Swedish Heart Lung Foundation, 20140131Swedish Cancer Society, 150815Swedish Diabetes AssociationSwedish Child Diabetes Foundation
Available from: 2017-02-20 Created: 2017-02-20 Last updated: 2018-01-13Bibliographically approved
Singh, K. (2017). Regulatory T cells in type 1 diabetes: the role of IL-35 in counteracting the disease. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Regulatory T cells in type 1 diabetes: the role of IL-35 in counteracting the disease
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Type 1 diabetes (T1D) is etiologically considered as an autoimmune disease, where insulin-producing β-cells are damaged by autoimmune attacks. Regulatory T (Treg) cells are immune homeostasis cells. In the present thesis I aimed to investigate the role of Treg cells and other immune cells in the early development of T1D. In order to do that, we first determined which immune cells that are altered at an early stage of the T1D development. We found that dendritic cells and plasmacytoid dendritic cells induce the initial immune response.

Next, we investigated the role of Treg cells in multiple low dose streptozotocin (MLDSTZ) induced T1D and in NOD mice. We found that the numbers of Treg cells were increased in both MLDSTZ and NOD mice when the MLDSTZ mice were hyperglycemic. However, the increased Treg cells showed a decreased production of anti-inflammatory cytokines (IL-10, IL-35 and TGF-β) and an increased expression of pro-inflammatory cytokines (IFN-γ and IL-17a). These results revealed that Treg cells switch their phenotype under T1D conditions.

IL-35 administration effectively prevented the development of, and reversed established MLDSTZ induced T1D. Treg cells from IL-35 treated mice showed an increased expression of the Eos transcription factor, accompanied by an increased expression of IL-35 and a decreased expression of IFN-γ and IL-17a. These data indicate that IL-35 administration counteracted the early development of T1D by maintaining the phenotype of the Treg cells. Furthermore, IL-35 administration reversed established T1D in the NOD mouse model by maintaining the phenotype of Treg cells, seemingly by inducing the expression of Eos. Moreover, the circulating level of IL-35 was significantly lowered in both new onset and long-standing T1D patients compared to healthy controls. In addition, patients with T1D with remaining C-peptide had significantly higher levels of IL-35 than patients lacking C-peptide, suggesting that IL-35 might prevent the loss of β-cell mass. In line with this hypothesis, we found that LADA patients had a higher proportion of IL-35+ tolerogenic antigen presenting cells than T1D patients.

Subsequently, we determined the proportions of IL-35+ Treg cells and IL-17a+ Treg cells in T1D patients with diabetic nephropathy (DN), which were age, sex and BMI matched with healthy controls and T1D patients. The proportion of IL-35+ Treg cells was decreased in DN and T1D patients, but IL-17a+ Treg cells were more abundant than in healthy controls. Furthermore, we found that the number of Foxp3+ Treg cells was increased in the kidneys of MLDSTZ mice. However, infiltration of mononuclear cells was seen in kidneys of these mice. In addition, kidney tissues of IL-35 treated MLDSTZ mice did not show any mononuclear cell infiltration. These results demonstrate that IL-35 may be used to prevent mononuclear cell infiltration in kidney diseases.

Our findings indicate that the numbers of Foxp3+ Treg cells are increased in T1D, but that these Treg cells fail to counteract the ongoing immune assault in islets and kidneys of hyperglycemic mice. This could be explained by a phenotypic shift of the Treg cells under hyperglycemic conditions. IL-35 administration reversed established T1D in two different animal models of T1D and prevented mononuclear cell infiltration in the kidneys by maintaining the phenotype of Treg cells.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. p. 51
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1373
Keywords
IL-35, regulatory T cells, type 1 diabetes, LADA, kidney and diabetic nephropathy
National Category
Medical and Health Sciences
Research subject
Medical Science; Immunology; Endocrinology and Diabetology
Identifiers
urn:nbn:se:uu:diva-329524 (URN)978-91-513-0084-9 (ISBN)
Public defence
2017-11-14, hall B42, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Available from: 2017-10-20 Created: 2017-09-18 Last updated: 2017-10-30
Lundin, S., Espes, D., Luo, Z., Blixt, M., Mejia Cordova, M., Carlsson, P.-O., . . . Singh, K. (2017). Role of regulatory B cells in clinical and experimental type 1 diabetes. Paper presented at 44th Annual Meeting of the Scandinavian-Society-for-Immunology (SSI), OCT 17-20, 2017, Stockholm, SWEDEN. Scandinavian Journal of Immunology, 86(4), 349-349
Open this publication in new window or tab >>Role of regulatory B cells in clinical and experimental type 1 diabetes
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2017 (English)In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 86, no 4, p. 349-349Article in journal, Meeting abstract (Other academic) Published
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-346972 (URN)000411865200233 ()
Conference
44th Annual Meeting of the Scandinavian-Society-for-Immunology (SSI), OCT 17-20, 2017, Stockholm, SWEDEN
Available from: 2018-03-28 Created: 2018-03-28 Last updated: 2018-03-28Bibliographically approved
Singh, K., Sandler, S. & Espes, D. (2017). The Increased Circulating Plasma Levels of Vascular Endothelial Growth Factor in Patients with Type 1 Diabetes Do Not Correlate to Metabolic Control. Journal of Diabetes Research, Article ID 6192896.
Open this publication in new window or tab >>The Increased Circulating Plasma Levels of Vascular Endothelial Growth Factor in Patients with Type 1 Diabetes Do Not Correlate to Metabolic Control
2017 (English)In: Journal of Diabetes Research, ISSN 2314-6745, E-ISSN 2314-6753, article id 6192896Article in journal (Refereed) Published
Abstract [en]

Aim. To characterize the plasma levels of vascular endothelial growth factor ( VEGF) in type 1 diabetes mellitus (T1D) and its relation to both present and historical metabolic control and microvascular complications.

Methods. Plasma levels of VEGF and routine clinical parameters were analyzed in 115 patients with long-standing T1D and 45 healthy controls (HC). All patients were under clinical routine diabetes treatment at Uppsala University Hospital.

Results. The plasma levels of VEGF were increased by 37% in patients with T1D when compared to HC (18.2 +/- 0.8 versus 13.2 +/- 1.0 pg/ml, p < 0.001). The levels of VEGF correlated to insulin needs and BMI but not to present or historical metabolic control. The levels of VEGF were similar in patients with T1D and microvascular complications (microalbuminuria and retinopathy) when compared with patients without microvascular complications. Historical HbA1c levels were found to be the best predictor for present metabolic control.

Conclusion. Circulating plasma levels of VEGF do not correlate to present or historical metabolic control in long-standing T1D and the levels are not affected by the presence of microvascular complications.

National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-359305 (URN)10.1155/2017/6192896 (DOI)000398400100001 ()28421206 (PubMedID)
Funder
Magnus Bergvall FoundationSwedish Child Diabetes Foundation
Available from: 2018-09-03 Created: 2018-09-03 Last updated: 2018-09-03Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-6771-7757

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