Maternal and Cord Blood 25-Hydroxyvitamin D3 Are Associated with Increased Cord Blood and Naive and Activated Regulatory T Cells: The Barwon Infant Study

Abstract CD4+CD25+ regulatory T cells (Tr) are negative regulators of immune responses. Studies of human Tr are restricted by their small numbers in peripheral blood and their hypoproliferative state. A recently established method achieved in vitro expansion and generation of Tr cell lines (Godfrey et al; Blood 2004,104:453-61). This approach facilitates the evaluation of cultured Tr cells as a novel form of immunosuppressive therapy and provides a system for molecular analysis of Tr. Activation of Ras and MAP kinases is mandatory for IL-2 production, viability and cell cycle progression of T cells. In anergic T cells activation of these signaling events is impaired, whereas activation of Rap1 is retained. Subsequently, anergic cells have defective IL-2 production, impaired cell cycle progression, and increased susceptibility to apoptosis. In the current study, we sought to determine the signaling and biochemical properties of Tr. Human CD4+CD25+ (Tr) and control CD4+CD25− (Tc) cell lines were generated from human cord blood cells. We examined activation of Ras, Rap1 and MAP kinases as well as cell cycle progression and cell viability, in response to TCR/CD3-plus-CD28 mediated stimulation. Stimulation was done for 15 min, 2 and 16 hrs for assessment of signaling events or for 24, 48 and 72 hrs for assessment of cell cycle progression and viability. Although activation of Rap1 was not affected, activation of Ras was reduced in Tr as compared to Tc. Activation of JNK and Erk1/2 MAP kinases was also significantly impaired. Both Tr and Tc entered the cell cycle and expressed cyclin E and cyclin A at 24 and 48 hrs of culture. However, p27 was downregulated only in Tc and not in Tr and hyperphosphorylation of Rb, which is the hallmark of cell cycle progression, was detected only in the Tc and not in the Tr population. At 72 hrs of culture, expression of cyclin E and cyclin A was dramatically diminished in Tr whereas it remained unchanged in Tc. More strikingly, expression of p27 in Tr was increased to levels higher than background. Since Tr do not produce IL-2, we examined whether addition of exogenous IL-2 would downregulate p27 and rescue Tr from defective cell cycle progression, similarly to its effect on anergic cells. Addition of exogenous IL-2 resulted in decrease of p27, sustained increase of cyclin E and cyclin A and cell cycle progression. Besides inhibiting cell cycle progression, p27 also promotes apoptosis. Therefore, we examined whether Tr had a higher susceptibility to apoptosis. As determined by Annexin V staining, Tr had a high degree of apoptosis only at 72 hrs of culture, when p27 expression was highly upregulated. Exogenous IL-2 reversed both p27 upregulation and apoptosis. Addition of IL-2 to Tr, also resulted in sustained IL-2-receptor-mediated activation of Erk1/2 at levels equivalent to those of Tc. Thus Tr cells share many biochemical and molecular characteristics of anergy, including defective TCR/CD3-plus-CD28-mediated activation of Ras and MAP kinases, increased expression of p27, defective cell cycle progression and high susceptibility to apoptosis. Moreover, these results suggest that TCR/CD3-mediated and IL-2 receptor-mediated signals converge at the level of MAP kinases to determine the fate of Tr cells towards expansion or cell cycle arrest and subsequent apoptosis.

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Expansion of Inhibitory NK Receptor (CD94/NKG2A)-Expressing Cytolytic CD8 T Cells and CD4+CD25+ Regulatory T Cells from the Same Cord Blood.

Blood ◽

10.1182/blood.v108.11.1739.1739 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1739-1739

Author(s):

Junji Tanaka ◽

Junichi Sugita ◽

Naoko Kato ◽

Tomomi Toubai ◽

Jun Ibata ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Cord Blood ◽

Cd8 T Cells ◽

Hla Class I ◽

Treg Cells ◽

Class I ◽

Target Cells ◽

Foxp3 Mrna ◽

Effector Cells

Abstract It has recently been shown that inhibitory natural killer cell receptors (NKRs) on not only NK cells but also on T cells negatively regulate NK cell and T cell functions through their binding to MHC class I molecules. The C-type lectin superfamily inhibitory NKR CD94/NKG2A heterodimer recognizes an HLA-E that preferably bound to a peptide derived from the signal sequences of most HLA class I. Therefore, CD94-expressing cells can monitor the global status of HLA class I on the tumor and leukemic cells and induce cytolytic attack without inhibitory signal against HLA class I decreased target cells resulting induction of graft-versus-leukemia (GVL) effect but does not attack normal cells with HLA class I expression resulting no enhancement of graft-versus-host disease (GVHD). On the other hand, CD4+ CD25+ regulatory T cells (Treg) contribute to suppress allogeneic immune responses and prevent transplant rejection and GVHD. In this study, we tried to expand CD94-expressing T cells and Treg cells from the same cord blood cells and then investigated their cytolytic characteristics and immunoregulatory function in order to develop a potential strategy of cell therapy for hematological malignancy. After CD4 enrichment by negative selection using magnetic cell sorting (MACS) (Miltenyi Biotec)(CD4-enriched fraction) from cord blood, CD4+ CD25+ cells were isolated by positive selection with anti-CD25 magnetic microbeads. We could get more than 1,000 fold expansion of CD94-expressing CD8 T cells from CD4-depleted fraction after 8 days culture with immobilized anti-CD3 monoclonal antibody (mAb) (1 μg/mL) and IL-15 (5 ng/mL). Isolated CD4+ CD25+ cells were cultured with anti-CD3/CD28 mAb-coated dynabeads and IL-15 (5 ng/mL) and we could get about 50 fold expansion of Treg cells for 8 days. These expanded Treg cells could suppress allogeneic mixed lymphocyte culture more than 80% (effector cells: Treg cells= 2:1) and expressed FoxP3 mRNA about 100 fold compared with isolated CD25-negative cells. Cytolytic activities of purified CD94-expressing cells (CD94 > 90%) detected by 4 hours 51Cr release assay against K562 were 68.8 ± 16.8 % (n=5). Coculture of CD94-expressing cells with expanded Treg cells (CD94-expressing cell: Treg cells= 1:1, preincubation 4 hours) did not have any effect on cytolytic activities of purified CD94-expressing cells against K562 cells (66.1 ± 19.8 %, n=5). CD94-expressing CD8 T cells with cytolytic activity could be expanded from CD4-deplted fractions and Treg cells with immunosuppressive activity and increased expression level of FoxP3 mRNA could be expanded from CD4-enriched fractions of the same cord blood. Expanded these cytolytic CD94-expressing CD8 cells might be able to induce GVL effect without enhancing GVHD and Treg cells might be able to suppress allogeneic response including GVHD and graft rejection. Therefore, this strategy may be useful to differentiate lymphocytes in cord blood to two different kinds of effector cells exhibiting cytolytic or immunoreguratoly characters.

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The Significance of mTOR Inhibitor, Everolimus in TGF-β-Induced Regulatory T cells from Cord Blood

Blood ◽

10.1182/blood.v118.21.2180.2180 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2180-2180

Author(s):

Tokiko Nagamura-Inoue ◽

Seiichiro Kobayashi ◽

Kazuo Ogami ◽

Yuki Yamamoto ◽

Kiyoko Izawa ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Cord Blood ◽

Cell Population ◽

Cd4 T Cells ◽

Mtor Inhibitor ◽

Ex Vivo ◽

Mtor Inhibitors ◽

Ex Vivo Expansion ◽

The Impact

Abstract Abstract 2180 Background: Regulatory T cells (Tregs) play an important role in immune-tolerance to allograft. Cord blood (CB) is rich in naïve T cells and is a promising source of inducible Tregs (iTregs), since it was reported that stable iTregs may be derived exclusively from naïve T cells. However, the standard method for iTregs has not yet been established. Here we studied the impact of mTOR inhibitors, rapamycin (Rap) and everolimus (Eve), on ex vivo expansion of iTregs from CB-CD4+ T cells. Methods: CB-CD4+ T cell were isolated using anti-CD4 monoclonal antibody (MAb)-conjugated magnetic beads, and cultured in a flask coated with anti-CD3/CD28 MAbs and supplemented with IL-2 and TGF-β in the presence or absence of Rap or Eve. After two weeks of culture, the total number of CD4+ T cells was calculated, and the incidence of CD25+Foxp3+ cell population among those was estimated by FACS. Results and Discussions: Both Rap and Eve significantly increased the incidence of CD25+Foxp3+ cell population in CD4+ T cells. However, Rap apparently inhibited their growth and did not increase the absolute number of CD25+Foxp3+ cells in comparison to the control. On the other hand, Eve contributed to efficient expansion of iTregs at the concentration between 1 and 50ng/ml without no significant inhibition of their growth. Expansion of CD4+ T cells with TGF-β and Eve yielded 71.5 ±23.5% purity of CD25+Foxp3+ cells which also expressed CTLA-4 as well as the memory phenotype, while the purity obtained with TGF-β only was 47.4±30.0% and that without TGF-β/Eve was 7.3±4.5%. Thus, an average of 2.95±2.8 x107 iTregs were obtained from the initial input of 5×104 CD4+ T cells. The resulting iTregs with TGF-β, TGF-β/Rap and TGF-β/Eve inhibited the proliferation of CFSE-labeled T cells stimulated with allogeneic dendritic cells. The precise mechanism for Foxp3 induction by mTOR inhibitors still remains to be elucidated. Furthermore, we found that expression of CD26 (DPP-IV) was significantly down-regulated in CD4+ T cells expanded with TGF-β and profoundly with TGF-β/Eve, while CD127 was negative after culture in all the conditions. Mean fluorescence intensity of CD26 indicated 67.5 in CD4+ T cells without TGF-β, 1.58 with TGF-β, 0.18 with TGF-β/Rap and 0.12 with TGF-β/Eve, respectively. Accordingly, CD26 negativity may be an indicator of iTregs together with Foxp3. Conclusion: mTOR inhibitor, Eve, is an efficient co-inducer of iTregs and applicable to ex vivo expansion of iTregs in a clinical setting. Disclosures: No relevant conflicts of interest to declare.

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Impaired function of regulatory T cells in cord blood of children of allergic mothers

Clinical & Experimental Immunology ◽

10.1111/j.1365-2249.2012.04630.x ◽

2012 ◽

Vol 170 (1) ◽

pp. 10-17 ◽

Cited By ~ 18

Author(s):

J. Hrdý ◽

I. Kocourková ◽

L. Prokešová

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Cord Blood ◽

Impaired Function

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11. Regulatory T-cells and cord blood

Biology of Blood and Marrow Transplantation ◽

10.1016/j.bbmt.2005.08.010 ◽

2005 ◽

Vol 11 (11) ◽

pp. 931-932

Author(s):

B.R. Blazar

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Cord Blood

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Adoptive Transfer of Umbilical Cord Blood-Derived Regulatory T Cells and Early Viral Reactivation

Biology of Blood and Marrow Transplantation ◽

10.1016/j.bbmt.2013.06.004 ◽

2013 ◽

Vol 19 (8) ◽

pp. 1271-1273 ◽

Cited By ~ 60

Author(s):

Claudio G. Brunstein ◽

Bruce R. Blazar ◽

Jeffrey S. Miller ◽

Qing Cao ◽

Keli L. Hippen ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Cord Blood ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Adoptive Transfer ◽

Viral Reactivation

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Third-party umbilical cord blood–derived regulatory T cells prevent xenogenic graft-versus-host disease

Cytotherapy ◽

10.1016/j.jcyt.2013.07.009 ◽

2014 ◽

Vol 16 (1) ◽

pp. 90-100 ◽

Cited By ~ 31

Author(s):

Simrit Parmar ◽

Xiaoying Liu ◽

Shawndeep S. Tung ◽

Simon N. Robinson ◽

Gabriel Rodriguez ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Cord Blood ◽

Umbilical Cord ◽

Graft Versus Host Disease ◽

Umbilical Cord Blood ◽

Third Party ◽

Host Disease ◽

Graft Versus Host

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Persistence of naive CD45RA+ regulatory T cells in adult life

Blood ◽

10.1182/blood-2005-06-2403 ◽

2006 ◽

Vol 107 (7) ◽

pp. 2830-2838 ◽

Cited By ~ 196

Author(s):

Nabila Seddiki ◽

Brigitte Santner-Nanan ◽

Stuart G. Tangye ◽

Stephen I. Alexander ◽

Michael Solomon ◽

...

Keyword(s):

T Cells ◽

Lymph Node ◽

T Cell ◽

Regulatory T Cells ◽

Cord Blood ◽

Peripheral Blood ◽

Cell Subset ◽

Human Infant ◽

Wide Range ◽

Adult Peripheral Blood

AbstractRegulatory T cells (TREGs) constitutively expressing CD4, CD25, and the transcription factor Foxp3 can prevent a wide range of experimental and spontaneous autoimmune diseases in mice. In humans, CD4+CD25bright T cells, predominantly within the CD45RO+ activated/memory subset in adults and the CD45RA+ naive T-cell subset in infants, are considered to be the equivalent subset. Using novel combinations of monoclonal antibodies (mAbs), we examined expression of CD25 in human infant thymus, cord blood, adult peripheral blood, lymph node, and spleen. In addition to the CD4+CD25bright T cells, subfractionation on the basis of CD45 splice variants indicated that all samples contained a second distinct population of cells expressing a slightly lower level of CD25. In adult peripheral blood, this population expressed a naive CD45RA+ phenotype. The corresponding population in lymph node, spleen, and cord blood showed some evidence of activation, and expressed markers characteristic of TREGs, such as cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). Sorted CD4+CD25+CD45RA+ T cells from both cord and adult blood expressed very high levels of mRNA for Foxp3 and manifested equivalent suppressive activity in vitro, indicating that they are bone fide members of the regulatory T-cell lineage. Targeting naive TREGs in adults may offer new means of preventing and treating autoimmune disease.

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Novel CD4+CD8+ Umbilical Cord Blood Regulatory T Cells

Blood ◽

10.1182/blood-2019-131948 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4446-4446

Author(s):

Meixian Huang ◽

Amit Verma ◽

Ke Zeng ◽

Mi-Ae Lyu ◽

Mitsutaka Nishimoto ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Cord Blood ◽

Research Funding ◽

P Value ◽

Q Value ◽

Double Positive ◽

Treg Population ◽

Equity Ownership ◽

Cytokine Assay

Background: Regulatory T cells (Treg), a well defined suppressor cell population with the phenotype of CD4+CD25+CD127- FoxP3+ are emerging as a promising adoptive therapy for a diverse subset of autoimmune diseases and inflammatory disorders. Specificlaly, cord blood (CB) Tregs have been shown to be superior to peripheral blood (PB) Tregs in their function and stability. Here, we define a novel subset of CB Tregs CD4+CD8+CD25+CD127- FoxP3+ that is not very well defined in the PB population and explore its' differentiation characteristics. Method: Tregs were isolated from CB or PB as described previously and cultured in the presence of CD3/28 beads and IL-2. On the day of harvest, cells were further sorted for CD8+ cell population and labelled as CD8+CD4+ (double positive CB Tregs: DP) and the leftover fraction as CD8-CD4+ (single positive CB Tregs: SP). Cells fractions were analyzed using flow cytometry and for cytokine assay, cells were activated (5 hours) with 1X Cell Stimulation Cocktail with Golgi inhibitors and stained for IFN-γ, IL-4, IL-17, IL-10 or TGFβ1 as per manufacturer's instructions. Additional cytokine assay was performed on the culture supernatants. DNA methylation assay was performed for the TSDR region as per manufacturer's instructions. Single cell RNAseq was performed at Albert Einstein Hospital and analyzed at Methodist Hospital. A threshold for original counts were applied to all four conditions, i.e. only transcripts with original counts>10 in all four. This step reduced the number of genes from 34626 to 16628; subsequently stringent cutoffs pairs showed two sets of genes- 1) Fold change>1.5, p-value<0.1, and 2) Fold change<2/3, p-value<0.1, this led to 73 rows, all with defined transcript names. Enrichment analysis using ConsensusPathDB for the above two gene sets were run to identify all pathways with at least one member-gene overlapping with either list, and the enrichment p-value for the pathway<0.1. Result: We cultured the Tregs isolated from CB or PB for 14 days and no evidence of a DP Tregs (0) was seen in PB as compared to a clearly defined DP Treg (8-26%) population in CB cultures. No differences were seen between the SP and DP CB Tregs in terms of the markers for mechanisms of suppression (FoxP3,Helios,CD39 ), homing (PSGL1, CLA, CD62L, CD49d), TGF-beta signaling (LAP, TGF-beta, GARP) and T cell signaling (LAG23, PD1, Icos, Tim3, CTLA-4, GITR). DP CBTregs consisted of a significantly higher expression of CCR4hiCXCR3hiCD95hipopulation. DP CB Tregs demonstrated a significantly higher IL-10 secretion compared to SP CB Tregs (Fig 1A) which correlated with the level of IL-10 secreted in the cell cuture supernatant (data not shown). While the degree of suppression of the proliferating conventional T cells was similar by the DP and SP CB Tregs (data not shown), a significantly higher demethylation of FoxP3 promoter and first intron region was observed in the DP vs SP CB Treg population (n=3) (Fig 1B). A total of 337 pathways were identified from Up-406 gene set, 80 of these pathways had enrichment p-value<0.05 and q-value<0.25 (Fig 1C). The Top 10 pathways enriched with members of Up-406 genes include (Fig 1D): TGF-β Receptor Signaling; Interleukin-10 signaling; Inflammatory bowel disease; IL12 signaling mediated by STAT4. A total of 158 pathways were identified from Down-314 gene set, 19 of these pathways had enrichment p-value<0.05 and q-value<0.25. The Top 10 pathways enriched with Down-314 genes included Immuno-regulatory interactions between a Lymphoid and a non-Lymphoid cell; ESC Pluripotency Pathways; Downstream signaling in naive CD8+ T cells; PDGF receptor signaling network; Nef Mediated CD8 Down-regulation (Fig E, F). Conclusion: We have identified a novel double positive CD4+CD8+ CB Treg population. Additional experiments for evaluating suppressor mechanism in addition to IL-10 secretion are ongoing. Figure 1 Disclosures Verma: Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria; BMS: Research Funding; Janssen: Research Funding. Iyer:Genentech/Roche: Research Funding; Incyte: Research Funding; Seattle Genetics, Inc.: Research Funding; Novartis: Research Funding; Arog: Research Funding; Bristol-Myers Squibb: Research Funding. Parmar:Cellenkos Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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