scholarly journals Immunosuppressive Diversity of Umbilical Cord-Derived Mesenchymal Stromal Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3584-3584
Author(s):  
Tokiko Nagamura-Inoue ◽  
Atsuko Takahashi ◽  
Yuki Yamamoto ◽  
Akiko Hori ◽  
Yuta Miharu ◽  
...  
Keyword(s):  
T Cells ◽  
Growth Factor ◽  
Regulatory T Cells ◽  
Umbilical Cord ◽  
Stromal Cells ◽  
Research Funding ◽  
Activated Lymphocytes ◽  
Hla Dr ◽  
Tnf Α ◽  
Ifn Γ

Recently, umbilical cord (UC) has become attracted source of mesenchymal stromal cells (MSCs) for immunotherapy such as treatment of severe acute graft versus host disease, because of abundant sources and ease of collection without invasive process. In previous reports on bone marrow-derived MSCs, there is diversity of immunosuppressive mechanisms of MSCs, which have not yet fully clarified in UC-MSCs. Here we studied the immunosuppressive properties of UC-MSCs on the activated immune system in vitro. Methods; UC was collected after obtaining informed consent from the mother. UC-MSCs were obtained from frozen-thawed UC tissue with explant method and expanded in culture medium. Mixed lymphocyte reaction (MLR) consisted with mononuclear cells (MNCs) stained with CFSE and allogeneic dendritic cell (DC) line (PMDC05) co-cultured with or without UC-MSCs was carried out, and the proliferation of CD4 and CD8-positive lymphocytes were analyzed by flowcytometry followed by FlowJo software. Migratory activities of UC-MSCs toward activated lymphocytes in MLR were evaluated by counting the migrating cells stained with DAPI through the insert filter under the microscope. The percentage of CD4+CD25+FOXP3+ regulatory T cells (Treg) before and after co-culture with UC-MSCs was analyzed by flowcytometry. Monocyte phenotypes co-cultured with UC-MSCs directly or separately by insert filter were analyzed with anti-CD14, CD206, and CD168 antibodies. Results: The UC-MSCs were positive for CD105, CD73, CD90, and negative for CD45 and HLA-DR. HLA-DR, CD80, CD86, and CD40 were negative even in the high concentration of IFN-γ, while BM-MSCs became positive for HLA-DR. PD-L2 was constitutively expressed in UC-MSC, while PD-L1 was induced upon the addition of IFN-γ. In MLR, responder T cell proliferation triggered by allogeneic DC was inhibited efficiently by 3rd party derived UC-MSCs. Mean+/- SD distribution index of proliferation in CD4- and CD8- positive cells co-cultured with UC-MSCs showed 7.6+/-4.4% and 8.8+/-3.9% compared to those without UC-MSCs (n=4, as different donor derived MSCs). This inhibition was attenuated by the addition of 1-Methyl-dl-tryptophan, Indoleamine 2, 3-dioxygenase (IDO-1) inhibitor, in a dose-dependent manner. The supernatant of MLR showed the increase of IFN-γ and TNF-α, which were also suppressed in MLR co-cultured with UC-MSCs. UC-MSCs migrated toward activated lymphocytes in MLR compared with those of non-activated lymphocytes significantly, which was inhibited by the addition of AG1296, known as platelet-derived growth factor inhibitor; and PPP as insulin-like growth factor (P<0.05). Cytokine beads array indicated the increase of RANTES, IL-8, and MIP-1. Regulatory T cells in CD4-positive T cells were significantly increased in the MNCs co-cultured with UC-MSCs. Interestingly, CD206-positive M2 monocytes were significantly increased in the MNCs cu-cultured with UC-MSC. Mean +/- SD percentage of CD206+CD80- M2 cells indicated 28.9+/-15.3% in MNCs and 83.3+/-11.2% in MNCs cu-cultured with UC-MSC (n=4, P<0.05). On the other hand, CD206-CD80+ M1 monocytes significantly increased in addition of LPS, which were inhibited by the co-culture with UC-MSCs (P<0.05). Furthermore IFN-γ and TNF-α secreted in the MNCs with LPS were suppressed by the co-culture of UC-MSCs. Discussions and Conclusion: These results demonstrated that UC-MSCs have the diverse immunosuppressive potency through migration toward the activated lymphocytes, suppressing activated T cells proliferation, regulatory T cells induction, and transition of monocyte phenotype. And UC-MSCs are the feasible and abundant source for immunotherapy. Disclosures Nagamura-Inoue: AMED: Research Funding. Nagamura:AMED: Research Funding. Tojo:AMED: Research Funding; Torii Pharmaceutical: Research Funding.

CD56+ human blood dendritic cells effectively promote TH1-type γδ T-cell responses

Blood ◽  
2009 ◽  
Vol 114 (20) ◽  
pp. 4422-4431 ◽  
Author(s):  
Georg Gruenbacher ◽  
Hubert Gander ◽  
Andrea Rahm ◽  
Walter Nussbaumer ◽  
Nikolaus Romani ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Human Blood ◽  
Γδ T Cells ◽  
Rapid Expansion ◽  
Γδ T Cell ◽  
Hla Dr ◽  
Tnf Α ◽  
Ifn Γ

Abstract CD56+ human dendritic cells (DCs) have recently been shown to differentiate from monocytes in response to GM-CSF and type 1 interferon in vitro. We show here that CD56+ cells freshly isolated from human peripheral blood contain a substantial subset of CD14+CD86+HLA-DR+ cells, which have the appearance of intermediate-sized lymphocytes but spontaneously differentiate into enlarged DC-like cells with substantially increased HLA-DR and CD86 expression or into fully mature CD83+ DCs in response to appropriate cytokines. Stimulation of CD56+ cells containing both DCs and abundant γδ T cells with zoledronate and interleukin-2 (IL-2) resulted in the rapid expansion of γδ T cells as well as in IFN-γ, TNF-α, and IL-1β but not in IL-4, IL-10, or IL-17 production. IFN-γ, TNF-α, and IL-1β production were almost completely abolished by depleting CD14+ cells from the CD56+ subset before stimulation. Likewise, depletion of CD14+ cells dramatically impaired γδ T-cell expansion. IFN-γ production could also be blocked by neutralizing the effects of endogenous IL-1β and TNF-α. Conversely, addition of recombinant IL-1β, TNF-α, or both further enhanced IFN-γ production and strongly up-regulated IL-6 production. Our data indicate that CD56+ DCs from human blood are capable of stimulating CD56+ γδ T cells, which may be harnessed for immunotherapy.

scholarly journals The Immunomodulatory Effect of Triptolide on Mesenchymal Stem Cells in Vitro

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5011-5011
Author(s):  
Haiping He ◽  
Atsuko Takahashi ◽  
Yuki Yamamoto ◽  
Akiko Hori ◽  
Yuta Miharu ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Complete Medium ◽  
Surface Markers ◽  
Rt Pcr ◽  
Tnf Α ◽  
Ifn Γ

Background: Mesenchymal stromal cells (MSC) are known to have the immunosuppressive ability and have been applied in clinic to treat acute graft-versus-host disease (GVHD), as one of severe complications after hematopoietic stem cells transplantation (HSCT) in Japan. However, MSC are activated to suppress the immune system only upon the stimulation of inflammatory cytokines and the clinical results of MSC therapies for acute GVHD are varied. It is ideal that MSC are primed to be activated and ready to suppress the immunity (=priming) before administration in vivo. Triptolide (TPL) is a diterpene triepoxide purified from a Chinese herb - Tripterygium Wilfordii Hook F (TWHF). It has been shown to possess anti-inflammatory and immunosuppressive properties in vitro. In this study, we aim to use TPL as the activator for umbilical cord-derived MSC (UC-MSC) to entry stronger immunosuppressive status. Methods: The proliferation of UC-MSC with TPL at the indicated concentrations for different time of 24, 48, 72, and 96 hours. Cell counting kit-8(CCK-8) was added in the culture medium to detect cell toxicity and the absorbance was measured using microplate reader. Flow cytometry was used to identify the MSC surface markers expression. TPL-primed UC-MSC were once replaced with fresh medium and co-culture with mixed lymphocyte reaction (MLR) consisted with mononuclear cells (MNCs) stained with CFSE and irradiated allogenic dendritic cell line (PMDC05) in RPMI 1640 medium supplemented with 10 % FBS (complete medium). IDO-1, SOD1, and TGF-β gene expression in TPL-primed UC-MSC and UC-MSC induced by 10 ng/ml IFN-γ and/or 15 ng/ml TNF-α were evaluated by RT-PCR. PDL1 and PDL2 expression in TPL-primed UC-MSC and UC-MSC in response to IFN-γ and/or TNF-α were checked by Flowjo. Results: Exposure of TPL for UC-MSC for 72hour at the concentration above 0.1 μM resulted in the cell damage significantly. Therefore, we added TPL in UC-MSC at 0.01μM of TPL for up to 48 hours, then washed thourouphly for the following culture for experiments. To evaluate the influence of TPL on the surface markers of UC-MSC, we cultured UC-MSC for 4 hours in complete medium following culture with 0.01μM of TPL for 20 hours (TPL-primed UC-MSC). TPL-primed UC-MSC revealed positive for CD105, CD73, and CD90, negative for CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface molecules as same as the non-primed UC-MSC. In MLR suppression by UC-MSC, the TPL-primed UC-MSC activity revealed stronger anti-proliferative effect on the CD4+ and CD8+ T cells activated by allogeneic DC than those of non-primed UC-MSC in MLR. Furthermore, the TPL-primed UC-MSC promoted the expression of IDO-1, SOD1 and TGF-β in response to IFN-γ+/-TNF-α by RT-PCR and enhanced the expression of PD-L1 by FACS analysis. Discussion:In this study, we found the TPL-primed UC-MSC showed stronger antiproliferative potency on CD4+ and CD8+ T cells compared with non-primed UC-MSC. TPL-primed UC-MSC promoted the expression of IDO-1, SOD1 and TGF-β stimulated by IFN-γ+/-TNF-α, although TPL alone did not induce these factors. Furthermore, we found that the PD1 ligand (PD-L1) was induced in TPL-primed UC-MSC, likely IFN-γ enhanced the PD-L1 expression, evaluated by flowcytometry. These results suggested that TPL-primed UC-MSC seemed more sensitive to be activated as the immunosuppressant. Here, we firstly report the new function of TPL to induce the upregulation of immunosuppressive effect, although the mechanisms of TPL inhibition to MSC need to be explore. Conclusively, TPL-primed UC-MSC might be applied for the immunosuppressive inducer of MSC. Figure Disclosures He: SASAGAWA Medical Scholarship: Research Funding; IMSUT Joint Research Project: Research Funding. Nagamura:AMED: Research Funding. Tojo:AMED: Research Funding; Torii Pharmaceutical: Research Funding. Nagamura-Inoue:AMED: Research Funding.

scholarly journals Expression of Regulatory T Cells in Jejunum, Colon, and Cervical and Mesenteric Lymph Nodes of Dogs Naturally Infected with Leishmania infantum

2014 ◽  
Vol 82 (9) ◽  
pp. 3704-3712 ◽  
Author(s):  
Maria M. Figueiredo ◽  
Beatriz Deoti ◽  
Izabela F. Amorim ◽  
Aldair J. W. Pinto ◽  
Andrea Moraes ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Lymph Nodes ◽  
Leishmania Infantum ◽  
Parasite Load ◽  
Mesenteric Lymph Nodes ◽  
Content Type ◽  
Mesenteric Lymph ◽  
Tnf Α ◽  
Ifn Γ

ABSTRACTUsing flow cytometry, we evaluated the frequencies of CD4+and CD8+T cells and Foxp3+regulatory T cells (Tregs) in mononuclear cells in the jejunum, colon, and cervical and mesenteric lymph nodes of dogs naturally infected withLeishmania infantumand in uninfected controls. All infected dogs showed chronic lymphadenitis and enteritis. Despite persistent parasite loads, no erosion or ulcers were evident in the epithelial mucosa. The colon harbored more parasites than the jejunum. Frequencies of total CD4+, total Foxp3, and CD4+Foxp3+cells were higher in the jejunum than in the colon. Despite negative enzyme-linked immunosorbent assay (ELISA) serum results for cytokines, levels of interleukin-10 (IL-10), gamma interferon (IFN-γ), transforming growth factor beta (TGF-β), and tumor necrosis factor alpha (TNF-α) were higher in the jejunum than in the colon for infected dogs. However, IL-4 levels were higher in the colon than in the jejunum for infected dogs. There was no observed correlation between clinical signs and histopathological changes or immunological and parasitological findings in the gastrointestinal tract (GIT) of canines with visceral leishmaniasis. However, distinct segments of the GIT presented different immunological and parasitological responses. The jejunum showed a lower parasite load, with increased frequencies and expression of CD4, Foxp3, and CD8 receptors and IL-10, TGF-β, IFN-γ, and TNF-α cytokines. The colon showed a higher parasite load, with increasing expression of IL-4.Leishmania infantuminfection increased expression of CD4, Foxp3, IL-10, TGF-β, IFN-γ, and TNF-α and reduced CD8 and IL-4 expression in both the jejunum and the colon.

Early Disease Relapse after Tyrosine Kinase Inhibitor Treatment Discontinuation in CML Is Related Both to Low Number and Impaired Function of NK-Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 812-812 ◽  
Author(s):  
Mette Matilda Ilander ◽  
Ulla Olsson-Strömberg ◽  
Hanna Lähteenmäki ◽  
Kasanen Tiina ◽  
Perttu Koskenvesa ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Treatment Discontinuation ◽  
Disease Relapse ◽  
Cell Counts ◽  
Tnf Α ◽  
Ifn Γ

Abstract Background: Recent reports suggest that approximately 40% of CML patients who have achieved sustained complete molecular remission are able to stop TKI treatment without disease relapse. However, there are no predictive markers for successful therapy discontinuation. Therefore, we set up an immunological sub-study in the ongoing pan-European EURO-SKI stopping study. Our aim was to identify predictive biomarkers for relapse/non-relapse and to understand more on the mechanisms of immune surveillance in CML. Methods: The EURO-SKI study started in 2012, and patients included were at least three years on TKI and at least one year in MR4 or deeper before the study entry. Basic lymphocyte immunophenotyping (the number of NK-, T- and B-cells) was performed at the time of therapy discontinuation and 1, 6, and 12 months after the TKI stop and in case of relapse (defined as loss of MMR, BCR-ABL1>0.1% IS). In addition, from a proportion of patients more detailed immunophenotypic and functional analyses (cytotoxicity of NK-cells and secretion of Th1 type of cytokines IFN-γ/TNF-α) were done at the same times. Results: Thus far 119 Nordic patients (imatinib n=105, dasatinib n=12, nilotinib n=2) who have discontinued TKI treatment within the EURO-SKI study have been included in the lymphocyte subclass analysis (results are presented from patients who have reached 6 months follow-up). Immunophenotyping analysis demonstrates that imatinib treated patients who were able to maintain remission for 6 months (n=36) had increased NK-cell counts (0.26 vs. 0.15x109cells/L, p=0.01, NK-cell proportion 18.9% vs. 11%, p=0.005) at the time of drug discontinuation compared to patients who relapsed early (before 5 months n=22). Furthermore, the phenotype of NK-cells was more cytotoxic (more CD57+ and CD16+cells and less CD62L+cells), and also their IFN-γ/TNF-α secretion was enhanced (19.2% vs. 13%, p=0.02). Surprisingly, patients who relapsed more slowly (after 5 months, n=16) had similar baseline NK-cell counts (0.37x109cells/L), NK-cell proportion (21.2%), and phenotype and function as patients, who were able to stay in remission. No differences in the NK-cell counts were observed between patients who had detectable or undetectable BCR-ABL1 transcripts at the baseline (0.22 x109cells/L vs. 0.31 x109cells/L, p=0.61). Interestingly, NK-cell count was higher in patients with low Sokal risk score than in patients with intermediate risk (0.33 x109cells/L vs. 0.20 x109cells/L, p=0.04). Furthermore, there was a trend that male patients had a higher proportion of NK-cells than females (21.6% vs. 15.7%, p=0.06). Pretreatment with IFN-α or the duration of imatinib treatment did not have an effect on NK-cell count or proportion. In comparison to the imatinib group, dasatinib treated patients had higher NK-cell counts at the baseline (median 0.52x109cells/L vs. 0.26x109cells/L, p=0.02), and also the proportion of CD27 (median 50% vs. 16%, p=0.01) and CD57 expressing (median 79% vs. 74%, p=0.05) NK-cells was higher. The follow-up time of dasatinib treated patients is not yet long enough to correlate the NK-cell counts with the success of the treatment discontinuation. The absolute number of T-cells or their function did not differ significantly between relapsing and non-relapsing patients at the time of treatment discontinuation. However, both CD4+ and CD8+ T-cells tended to be more mature in patients who stayed in remission compared to patients who relapsed early (CD4+CD57+CD62L- median 5.7% vs. 2.4%, p=0.06, CD8+CD62L+CD45RA+ 13% vs. 26.7%, p=0.05). The analysis of follow-up samples showed that in patients who stayed in remission the Th1 type cytokine (IFN-γ/TNF-α) secretion of CD8+T-cells increased at 6 months compared to baseline (23.6 vs. 18.5%, p=0.07). Same phenomenon was observed in the late relapsing group at relapse compared to baseline (37.9 vs. 13.5%, p=0.03). No similar increase was observed in the early relapsing group. Conclusions: Low NK-cell numbers and poor cytokine secretion may predict early disease relapse after TKI discontinuation. However, patients who relapse later have high numbers of normally functioning NK-cells. Further research (detailed phenotypic analysis of NK- and T-cells including activating and inhibitory receptors and immune checkpoint molecules) and correlation of biomarker data with clinical parameters are ongoing to understand the ultimate determining factors of relapse. Disclosures Själander: Novartis: Honoraria. Hjorth-Hansen:Novartis: Honoraria; Bristol-myers Squibb: Honoraria; Ariad: Honoraria; Pfizer: Honoraria. Porkka:BMS: Honoraria; BMS: Research Funding; Novartis: Honoraria; Novartis: Research Funding; Pfizer: Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

scholarly journals Mechanisms of Resistance and Determinants of Response of the GPRC5D-Targeting T-Cell Redirecting Bispecific Antibody JNJ-7564 in Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 8-9
Author(s):  
Christie P.M. Verkleij ◽  
Marloes Broekmans ◽  
Amy Wong ◽  
Sonja Zweegman ◽  
Raluca Verona ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Bispecific Antibody ◽  
Cell Lysis ◽  
Advisory Committees ◽  
Hla Dr ◽  
Tnf Α ◽  
The Impact

Introduction: New immunotherapies directed against CD38, SLAMF7 or BCMA have significantly improved the outcome of multiple myeloma (MM) patients. However, most patients eventually relapse, underscoring the need for additional immunotherapeutic targets. We have previously shown that expression levels of GPRC5D, an orphan G protein-coupled receptor, are significantly higher on MM cells, compared to normal plasma cells or other immune cells. We also showed that the novel GPRC5DxCD3 bispecific antibody (BsAb) JNJ-7564, has promising anti-MM activity in patient-derived BM samples (Verkleij et al., EHA 2019). To elucidate which factors contribute to the observed heterogeneity in ex vivo response, we analyzed the impact of tumor and patient characteristics on efficacy of JNJ-7564. We further investigated whether tumor-intrinsic factors may be determinants of response by also testing in these assays JNJ-7957, a BCMA-targeting BsAb that differs from JNJ-7564 only in the tumor-antigen-binding domain. Methods: Bone marrow (BM) samples obtained from 13 newly diagnosed (ND), 17 daratumumab-naive relapsed/refractory (DARA-naive RR; median of 3 prior therapies) and 15 daratumumab-refractory (DARA-R, median of 6 prior therapies) MM patients were analyzed for tumor- and immune cell composition, and subsequently incubated with JNJ-7564 (0.00128-4.0 µg/mL) or JNJ-7957 (0.8 µg/mL). After 48 hours, MM cell lysis was assessed by flow cytometry. Luciferase-transduced MM cell lines were incubated with JNJ-7564 (0.032-4.0 µg/mL) in the presence of healthy peripheral blood mononuclear cells (PBMCs), purified CD4+CD25- T-cells or regulatory T-cells (Tregs). After 48 hours, MM cell lysis was assessed by bioluminescence assay. Results: We found no difference in JNJ-7564 efficacy with respect to disease stage (NDMM vs DARA-naive RRMM vs DARA-R MM, P=0.48). Importantly, the presence of high-risk cytogenetic abnormalities [del(17p), t(4;14) and t(14;16)] did not impair JNJ-7564 efficacy. The level of target expression was an important determinant of response, as evidenced by superior MM cell lysis in samples with higher than median GPRC5D expression, when compared to lower GPRC5D expression (Fig. 1A). Inferior MM cell lysis was observed in older patients (>67 years), in samples with low T-cell counts or low effector:target (E:T) ratios, and in those with a high frequency of PD-1+ T-cells, HLA-DR+ activated T-cells, or Tregs. These determinants of response also affected JNJ-7564-mediated T-cell activation and degranulation. To further analyze the impact of Tregs, we performed additional cell line experiments. Purified Tregs impaired T-cell proliferation, and were significantly less potent to kill MM cells when redirected by JNJ-7564, compared to CD4+CD25- T-cells (Fig. 1B). This was accompanied by reduced secretion of IFN-γ, TNF-α, IL-2 and granzyme B. To evaluate the impact of BM stromal cells (BMSCs) on JNJ-7564 activity, MM cell lines were co-incubated with PBMCs and patient-derived BMSCs. Direct cell-cell contact hampered MM cell lysis, while indirect contact (transwell) did not affect JNJ-7564 activity. Direct contact also decreased secretion of TNF-α and IL-2, and reduced GPRC5D expression on MM cells, contributing to BMSC-mediated resistance to JNJ-7564. Finally, we simultaneously evaluated the single agent activity of both JNJ-7564 and JNJ-7957 (0.8 µg/mL, dose whereby a plateau in MM cell lysis was observed with both BsAbs) in 40 BM samples. MM cell lysis induced by both agents was strongly correlated (Fig. 1C). In 6 samples, both agents exhibited poor activity (<45% lysis), whereas in 9 samples very good activity was observed (>80% lysis). Comparison of characteristics between these groups showed that a low E:T ratio (Fig. 1D) and high frequency of Tregs (Fig. 1E) significantly impaired efficacy of both BsAbs, suggesting patient-specific factors can determine response to T-cell redirectors targeting different antigens. Conclusion: We show that tumor-related factors, such as GPRC5D expression, as well as differences in the composition of the BM microenvironment, including E:T ratio, frequency of PD-1+ or HLA-DR+ T-cells or immune-suppressing Tregs or BMSCs, contribute to the variability in response to JNJ-7564. Our data indicate that strategies aiming at optimizing E:T ratio (e.g. induction therapy) or Treg depletion, may improve response to T-cell redirecting antibodies in MM. Disclosures Wong: Jhonson & Jhonson: Current Employment. Zweegman:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees. Verona:Johnson & Johnson: Current Employment, Current equity holder in publicly-traded company. Adams:Johnson & Johnson: Ended employment in the past 24 months. Mutis:Janssen Pharmaceuticals: Research Funding; Genmab: Research Funding; Takeda: Research Funding; Onkimmune: Research Funding; Gadeta: Research Funding. van de Donk:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Ferrer: Membership on an entity's Board of Directors or advisory committees.

Maturation and Phenotypic Diversity of Human CD4+ Regulatory T Cells in Umbilical Cord Blood and Peripheral Blood from Healthy Donors

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2357-2357
Author(s):  
Tiago R Matos ◽  
Hongye Liu ◽  
Masahiro Hirakawa ◽  
Ana Cristina Alho ◽  
Jerome Ritz
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Functional Markers ◽  
Large Set ◽  
Apoptotic Markers ◽  
Hla Dr ◽  
Terminal Effector

Abstract Introduction: CD4+ FoxP3+ CD25+ regulatory T cells (Treg) are required for maintenance of immune tolerance and immune homeostasis. Quantitative or functional Treg deficiency has been correlated with autoimmune disease, allergy, allograft rejection and graft versus host disease. Conversely, increased Treg can suppress tumor immunity resulting in tumor progression. Treg express a large number of cellular markers that reflect their level of maturation, functionality, activation and migratory capacity. Nevertheless, it has not previously been possible to integrate the expression of these various markers and correlate their expression with human Treg differentiation in vivo. Methods: We used single cell mass cytometry (CyTOF) to simultaneously study 36 phenotypic and functional markers of human Treg in samples obtained from umbilical cord blood (CB) (n=4) and healthy adult donors (n=10). Wanderlust trajectory detection algorithm was used to analyze temporal positioning of Treg across development. To quantify Treg heterogeneity we used ACCENSE; an analysis tool that combines a nonlinear dimensionality reduction algorithm (t-SNE) with spectral clustering algorithm and automated cell classification into subpopulations. Results: Using Wanderlust to characterize Treg maturation, the majority of CB Treg were naive (CD45RA+) and CB memory Treg (CD45RA-) were poorly differentiated with minimal expression of activation (HLA-DR) and pro-apoptotic (CD95) markers (Figure 1A). Adult Treg contained few naive cells and mature Treg effector cells expressed high levels of activation and pro-apoptotic markers. (Figure 1B). Using Wanderlust together with spearman correlation, 5 discrete stages of Treg maturation were identified; 1) recent thymic emigrants (RTE), 2) naive, 3) effector, 4) activated and 5) terminal effector. RTE Treg defined by expression of CD45RA, CD31 and CCR7, also expressed markers of proliferation (KI67) and functionality (Tbet, PDL-1, Helios) at low levels but lacked functional CTLA4 and TIM-3. Naive Treg lacked expression of CD31 but expressed other markers characteristic of RTE. Effector Treg expressed increased levels of CD95, CTLA-4, CCR7, GITR, Helios and FoxP3 but lacked HLA-DR. Activated effector Treg expressed the highest levels of FoxP3, Helios and Ki67, along with functional markers (CD28, CXCR3, ICOS, GITR, CD39, CTLA-4, TIM-3) and homing molecules (vascular endothelial CCR5, gut addressin ACT-1, skin addressins CCR4, CLA). Activated Treg express the highest levels and diversity of functional markers along with the ability to migrate to different tissues. Lastly, terminal effector Treg express a more restricted set of functional and homing markers (CD28, CTLA-4, ICOS and CCR5) with less diversity. Markers of exhaustion (PD-1 and TIM-3) are also expressed by effector, activated and terminal effector Treg. Pro-apoptotic markers (CD95high BCL2low) are primarily expressed by activated and terminal effector Treg. Using ACCENSE to evaluate Treg diversity allowed further identification of discrete Treg sub-populations within each maturation state. RTE and naive Treg appear very homogeneous and appear as a single cluster in both CB and adults. In contrast, effector, activated and terminal effector Treg are more heterogeneous and are visualized as 9 distinct clusters (Figure 1C, D). This clustering reflects distinct subsets of memory Treg that co-express various combinations of functional markers in our panel. All 9 Treg effector populations are present in CB, but at much lower levels. Treg effector cell diversity is maintained but does not increase as Treg mature and expand in adults and RTE/naive Treg become less prevalent. Conclusion: Our study is the first to quantify human Treg heterogeneity based on expression of a large set of activation, proliferation, tissue homing and functional markers in conjunction with stages of Treg maturation and differentiation. These studies define 5 stages of Treg maturation and 10 clusters of Treg diversity based on differential expression of phenotypic and functional markers. Similar approaches can be applied to describe maturation and diversity of other cell populations. Further application of this CyTOF panel can be used to study Treg maturation and diversity after hematopoietic stem cell transplantation and in immune and inflammatory diseases, to identify specific defects that may contribute to immune dysfunction. Disclosures No relevant conflicts of interest to declare.

scholarly journals Expression of Regulatory γδ T Cells in Patients with Acute Graft-Versus-Host Disease

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5784-5784
Author(s):  
Li Xuan ◽  
Li Gao ◽  
Xiuli Wu ◽  
Zhiping Fan ◽  
Fen Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Effective Treatment ◽  
Research Funding ◽  
Γδ T Cells ◽  
Guangdong Province ◽  
Expression Levels ◽  
Graft Versus Host ◽  
Tnf Α ◽  
Ifn Γ ◽  
Acute Graft

Abstract Background Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the most effective therapy for hematologic malignancies. Acute graft-versus-host disease (aGVHD) is a major complication after allo-HSCT and leads to high transplant-related mortality. Regulatory γδ T cells (γδ Tregs), as a novel subset of γδ T cells with Foxp3 expression and immunosuppressive effect, are found to effectively alleviate aGVHD in mice model. However, the effect of γδ Tregs on the occurrence and development of aGVHD are not fully understood. The aim of this study is to investigate the expression levels and clinical significance of γδ Tregs in patients with aGVHD. Methods The immunophenotyping of γδ Tregs was analyzed in peripheral blood from 15 patients with aGVHD at the following two time points (at the onset of aGVHD and two weeks after the treatment), using flow cytometry. Twelve patients responded effectively to the treatment within two weeks, and 3 did not respond within two weeks. The expression levels of immunoregulatory-associated molecules (Foxp3, CD25, CTLA-4, GITR, TLR8, RORc, STAT-1 and STAT-3) were analyzed in peripheral blood, using real-time RT-PCR with SYBR GreenⅠstaining. Liquichip technology was used to analyze the concentration of interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-10, IL-17 and tumour necrosis factor-α (TNF-α) in cultural supernatant. Results The proportions of γδ T, Vδ1 T and Vδ2 T cells did not differ significantly between the untreated patients and those with effective treatment (P=0.308, P=0.117 and P=0.099). However, the proportions of Foxp3+γδ T, Foxp3+Vδ1 T and Foxp3+Vδ2 T cells were significantly increased after effective treatment (P=0.023, P=0.008 and P=0.028). The mRNA expression levels of Foxp3, RORc and TLR8 genes were significantly increased after effective treatment (P=0.060, P=0.001 and P=0.041). The expression levels of CD25, CTLA-4, GITR, STAT-1 and STAT-3 genes were similar between the untreated patients and those with effective treatment (P=0.95, P=0.421, P=0.605, P=0.186 and P=0.809). In addition, the concentration of IL-10 was decreased after effective treatment (P=0.005), and the concentration of IFN-γ, IL-4, IL-17 and TNF-α was similar between the two groups (P=0.662, P=0.314, P=0.152 and P=0.254). At the same time, we found that the proportion of Foxp3+γδ T cells and the expression level of Foxp3 gene both had a decreased tendency after ineffective treatment. The concentration of IL-10 had a increased tendency after ineffective treatment. Conclusions γδ Tregs might influence in the occurrence and development of aGVHD. Disclosures Liu: Science and technology planning project of Guangdong Province (2014B020226004);: Research Funding; the project of health collaborative innovation of Guangzhou city (201400000003-1, 201400000003-4, 201508020254).: Research Funding; National Natural Science Foundation of China (81270647, 81300445, 81470349, 81400141, U1401221, 81401315).: Research Funding; Natural Science Foundation of Guangdong Province (2014A030310171, 2016A030310390) ;: Research Funding.

scholarly journals Adoptive Cord Blood T Regulatory Cell Therapy Leads to Resolution of Inflammation and Decreased Proteinuria in Lupus Nephritis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 5-6
Author(s):  
Mi-Ae Lyu ◽  
Joseph D. Khoury ◽  
Meixian Huang ◽  
Mitsutaka Nishimoto ◽  
Ke Zeng ◽  
...  
Keyword(s):  
T Cells ◽  
Lupus Nephritis ◽  
Cell Therapy ◽  
Cord Blood ◽  
Research Funding ◽  
Private Company ◽  
Adoptive Therapy ◽  
Post Transplant ◽  
Tnf Α ◽  
Ifn Γ

Background. Systemic lupus erythematosus (SLE) is associated with widespread inflammation with multi-organ involvement where renal failure is the most dreaded and fatal complication. Adoptive therapy with cord blood (CB) derived T regulatory cells has been shown to improve outcomes in disease driven by inflammation including graft versus host disease (GvHD), inflammatory bone marrow disorder, COVID-19 induced acute respiratory distress syndrome. We hypothesize that CB Treg therapy can treat lupus nephritis. Methods. The suppressive abilities of CB Tregs expressing CD4+CD25highCD127lowFoxP3+ were assayed by human cytokines assay kits (IL-10, IFN-γ, IP-10, TNF-α, IL-6, and IL-17A) in the cell culture supernatants. For examining the efficacy of CB Tregs in vivo, SLE xenograft model was created with female Rag2-/-γc-/- mice transplanted with 3x106 human SLE-peripheral blood mononuclear cells (PBMCs) by intravenous injection on day 0. The mice were allowed to develop disease and on day 30 post-transplant, they were divided into 2 groups: i) control and ii) treatment. 1x107 ex vivo-expanded, cryopreserved, allogeneic, non-HLA matchedCB Tregs were injected into SLE xenografts intravenously once per week for 4 weeks through the tail vein. Serial blood draws were performed for the phenotypic analysis, cytokine assay and anti-double stranded (ds)DNA IgG antibody analysis. Serial examination of the urine samples was performed for creatinine and albumin quantification. Histopathologic examination of the harvested organs was performed at the time of planned euthanasia at 13 weeks. Results. Co-culture of CB Tregs with the pathogenic SLE-PBMCs decreased the secretion of inflammatory cytokines including IFN-γ, IP-10, TNF-α, IL-6, and IL-17A (Figure A) with a reciprocal increase in the secretion of the anti-inflammatory IL-10 cytokine (Figure B). Adoptive therapy with CB Treg cells led to a significant decrease in circulating CD8+ effector T cells and an increase in CD4+ helper T cells (Figure C). CB Treg recipients showed preserved weight gain (Figure D), lower GvHD score (Figure E) and improved overall survival (Figure F). A significant decrease in proteinuria at 9 weeks post-transplant (Figure G) correlated with a decrease in anti-dsDNA IgG Ab levels (Figure H) and soluble CD40 ligand levels (Figure I). Histopathological results from two index cases from each arm (Figure J) demonstrated that CB Treg recipients show reduced T-cells (CD3+) (Figure K) and B-cells (CD20+) (Figure L) in the kidneys, as well as a decrease in the lymphoid infiltration into glomeruli and renal parenchyma as compared to the control arm (Figure M). Conclusion. We are the first to demonstrate the benefit of allogeneic CB Treg cell therapy for treatment of lupus nephritis. We propose to examine such a strategy in the clinical setting. Figure Disclosures Nishimoto: Janssen Pharmaceutical K.K.:: Research Funding; Bayer Yakuhin, Ltd:: Research Funding. Sadeghi:Cellenkos Inc.: Current Employment. Parmar:Cellenkos Inc.: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

scholarly journals Activation Phenotype of Mycobacterium tuberculosis-Specific CD4+ T Cells Promoting the Discrimination Between Active Tuberculosis and Latent Tuberculosis Infection

2021 ◽  
Vol 12 ◽  
Author(s):  
Ying Luo ◽  
Ying Xue ◽  
Liyan Mao ◽  
Qun Lin ◽  
Guoxing Tang ◽  
...  
Keyword(s):  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
Latent Tuberculosis Infection ◽  
Latent Tuberculosis ◽  
Active Tuberculosis ◽  
Tuberculosis Infection ◽  
Antigen Stimulation ◽  
Hla Dr ◽  
Tnf Α ◽  
Ifn Γ

BackgroundRapid and effective discrimination between active tuberculosis (ATB) and latent tuberculosis infection (LTBI) remains a challenge. There is an urgent need for developing practical and affordable approaches targeting this issue.MethodsParticipants with ATB and LTBI were recruited at Tongji Hospital (Qiaokou cohort) and Sino-French New City Hospital (Caidian cohort) based on positive T-SPOT results from June 2020 to January 2021. The expression of activation markers including HLA-DR, CD38, CD69, and CD25 was examined on Mycobacterium tuberculosis (MTB)-specific CD4+ T cells defined by IFN-γ, TNF-α, and IL-2 expression upon MTB antigen stimulation.ResultsA total of 90 (40 ATB and 50 LTBI) and another 64 (29 ATB and 35 LTBI) subjects were recruited from the Qiaokou cohort and Caidian cohort, respectively. The expression patterns of Th1 cytokines including IFN-γ, TNF-α, and IL-2 upon MTB antigen stimulation could not differentiate ATB patients from LTBI individuals well. However, both HLA-DR and CD38 on MTB-specific cells showed discriminatory value in distinguishing between ATB patients and LTBI individuals. As for developing a single candidate biomarker, HLA-DR had the advantage over CD38. Moreover, HLA-DR on TNF-α+ or IL-2+ cells had superiority over that on IFN-γ+ cells in differentiating ATB patients from LTBI individuals. Besides, HLA-DR on MTB-specific cells defined by multiple cytokine co-expression had a higher ability to discriminate patients with ATB from LTBI individuals than that of MTB-specific cells defined by one kind of cytokine expression. Specially, HLA-DR on TNF-α+IL-2+ cells produced an AUC of 0.901 (95% CI, 0.833–0.969), with a sensitivity of 93.75% (95% CI, 79.85–98.27%) and specificity of 72.97% (95% CI, 57.02–84.60%) as a threshold of 44% was used. Furthermore, the performance of HLA-DR on TNF-α+IL-2+ cells for differential diagnosis was obtained with validation cohort data: 90.91% (95% CI, 72.19–97.47%) sensitivity and 68.97% (95% CI, 50.77–82.73%) specificity.ConclusionsWe demonstrated that HLA-DR on MTB-specific cells was a potentially useful biomarker for accurate discrimination between ATB and LTBI.

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