Nina Nagel is gratefully acknowledged for technical assistance during the video shoot and experimental preparation. We thank Eva-Maria Weiss for help with the intracellular FOXP3 staining protocol and Elisabeth Suri-Payer and Nina Oberle for establishing the nTreg isolation protocol. Matilda Eriksson and Peri Noori are acknowledged for laboratory management. 
Funding: A.S. was supported by a Marie Curie Intra European Fellowship within the 7th European Community Framework Programme, the Dr. &#197;ke Olsson Foundation and KI research foundations; A.S. and J.T. were supported by a CERIC (Center of Excellence for Research on Inflammation and Cardiovascular disease) grant, J.T. was supported by Vetenskapsr&#229;det Medicine and Health (Dnr 2011-3264), Torsten S&#246;derberg Foundation, FP7 STATegra, AFA Insurance and Stockholm County Council.

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The authors have nothing to disclose.

기술 된 프로토콜은 인간의 CD4 + Foxp3의 + 인간의 순진한 CD4 + T 세포에서 iTregs의 강력한 유도 할 수 있습니다. 우리가 우수한 시험 관내 억제 기능 22 iTregs의 유도, TGF-β, ATRA 및 라파 마이신의 조합을 사용하여, 최근에 기술 된 새로운 프로토콜을 포함한다. 게시 된 다른 프로토콜들에 비해 다른 이점은 단독 -2- IL의 존재 하에서 활성화 된 대조군 세포와 함께 특정 iTreg 유발 요...

CD4 + CD25 + + Foxp3를 조절 T 세포 (Tregs)는 다른 면역 세포를 억제 말초 관용의 중요한 매개체 방지자가 면역 과도한 염증 일이다. Tregs의 중요성은, 중증 전신성자가 면역 질환에 이르게하는 Tregs의 손실로 인해`master&#39; TREG 전사 인자 forkhead 상자 P3의 돌연변이로 인간의 질병 immunodysregulation의 polyendocrinopathy의 병증의 X 연결된 증후군 (IPEX), (Foxp3를)을들 수있다 어린 나이에 치명적. 그러나 Tregs는 또한 특정 설정이있는 항 종양 면역을 방해 할 수있는 면역 체계의 양날의 검으로 작용한다. TREG 번호와 기능의 치료 조작은 수많은 임상 연구에 따라서 달라질 수 있습니다. 암에서 Tregs의 고갈은 바람직 할 수 있으며, 임상 적 접근 방법의 일부 성공은 추가 연구 (3)를 권장합니다. 세비에서 Tregs의 치료 효과뿐만 아니라자가 면역 및 염증 질환에RAL 마우스 질병 모델, 입양 TREG 전송의 최근 처음에 남자 임상 시험 그라프 대 -host 질환 (GVHD) 4 방지 - 7 매우 유망한 결과를 보여 주었다 제 1 형 당뇨병 (8) 치료에 안전성을 평가하기 위해. 11 - 자연 Tregs (nTregs가) 건강 (9)을 유지하는 비 중복 필수 기능과 흉선 유래 tTregs 및 주변으로 유도 pTregs를 포함 발생. 그러나 nTreg 번호는 순진한 T 세포 전구체 (12)으로부터 체외에서 유도 Tregs (iTregs)의 보완적인 접근 방식을 장려, 제한됩니다. 아마도 인해 Foxp3의 유전자 좌위 (13)의 소위 TREG 별 탈 메틸화 지역 (TSDR)에서 탈 메틸화 부족 iTregs의 또 안정성, 우려 남아 있고 여러 연구는 Tregs 유도 생체 내에서 14 더 안정되어 있음을 나타냅니다. 지금까지 Foxp3의 최고의 단백질 m 남아인간 기존의 CD4 + CD25- T 세포가 일시적으로 활성화 (15, 16)에 Foxp3의 중간 수준을 표현하기 때문에 Tregs에 대한 arker하지만 그것은 절대적으로 특정하지 않습니다. 상당한 진전은 Foxp3를 발현 조절되는지 설명 하였지만, 더 특히 인간 세포에서의 유도 Foxp3의 안정성 및 기능에 대하여 발견되고 유지된다. nTregs에 차이에도 불구하고, 시험 관내에서 Foxp3를 유도 + CD4 + T 세포는 Foxp3를 유도 분자 메커니즘을 연구하기위한 모델 시스템으로 사용할 수있는 더 유사한 iTregs의 생성을 허용 장래 프로토콜을 개발하는 시작점 미래의 입양 전송 전략에 적용 할 수있는 생성 Tregs를 생체. 거기에 인간 iTregs 유도 할`금 standard&#39; 프로토콜은없고, 현재 프로토콜 생체 TREG 유도 조건을 모방에 기초하여 개발되었다 : 2 (IL-2 인터루킨) 및 변형 성장 인자 β (TGF-β) 시그널링은 생체 17 Foxp3의 유도에 매우 중요하고, 올 - 트랜스 레티노 산 (ATRA) - 창자 연관된 수지상 세포에 의해 생체 내에서 생성되는 - 자주 Foxp3의 유도를 향상시키는 데 사용된다 21-18을 체외있다. 우리는 부티레이트 (22), 최근에 쥐 TREG 유도 (23, 24)을 증가시키기 위해 도시 된 장내 미생물 유래 단쇄 지방산을 사용하여 추가 인간 TREG 유도 프로토콜을 개발했습니다. 또한, 최근 그을 촉진하는 것으로 알려져 억제제 후자는 라파 마이신 (mTOR의)의 임상 적으로 승인 된 포유류 대상인, TGF-β, ATRA의 조합을 사용하여, 22 라파 마이신에 의해 시험관 내에서 우수한 억제 기능 iTregs의 생성을위한 새로운 프로토콜을 확립 인간의 TREG 확장 25, 26시 Foxp3의 유지 보수. 이 방법의 재현성을 설명합니다인간 CD4 + Foxp3의 + 상이한 조건들의 세트를 이용 iTregs 및 유세포 정량 역전사 중합 효소 연쇄 반응에 의해 그 이후의 표현형의 체외 세대 (QRT-PCR)은 Foxp3를 발현하고, 다른 TREG 서명 분자 등을의 프로토콜 별 무늬를 공개 CD25, CTLA-4, EOS뿐만 아니라, IFN-γ 및 SATB1 식 (22)의 억압 등. 생성 된 세포 집단은 일반적 Foxp3의 규제에 관한 또는 부티르산 또는 라파 마이신과 같은 특정 화합물은 특정 효과를 연구하거나, 억제 활성에 대한 기능 분석을 위해 또는 분자 연구에 사용될 수있다. TREG 차별화를 운전하는 분자 메커니즘의 추가 이해는 특히 TREG 생성과 기능에 관여하는 분자를 대상으로자가 면역 암에서 미래의 치료 접근에 매우 관련이있다.

<table><tbody><tr><td>All-trans retinoic acid</td><td>Sigma Aldrich</td><td>R2625-50MG&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>anit-human Foxp3-APC clone 236A/E7</td><td>eBioscience</td><td>17-4777-42</td><td></td></tr><tr><td>anti-human CD25 microbeads</td><td>Miltenyi Biotec</td><td>130-092-983</td><td></td></tr><tr><td>anti-human CD25-PE</td><td>Miltenyi Biotec</td><td>130-091-024</td><td></td></tr><tr><td>anti-human CD28 antibody, LEAF Purified&amp;nbsp;</td><td>Biolegend</td><td>302914</td><td></td></tr><tr><td>anti-human CD3 Antibody, LEAF Purified&amp;nbsp;</td><td>Biolegend</td><td>317315</td><td></td></tr><tr><td>anti-human CD45RA, FITC</td><td>Miltenyi Biotec</td><td>130-092-247</td><td></td></tr><tr><td>anti-human CD45RO PE clone UCHL1</td><td>BD Biosciences</td><td>555493</td><td></td></tr><tr><td>anti-human CD4-PerCP clone SK3; mIgG1</td><td>BD Biosciences</td><td>345770</td><td></td></tr><tr><td>anti-human CD8-eFluor 450 (clone OKT8), mIgG2a</td><td>eBioscience</td><td>48-0086-42&amp;nbsp;</td><td></td></tr><tr><td>anti-human CTLA-4 (CD152), clone BNI3, mIgG2ak, Brilliant violet 421</td><td>BD Biosciences</td><td>562743</td><td></td></tr><tr><td>anti-human IFN-g FITC clone 4S.B3; mIgG1k</td><td>eBioscience</td><td>11-7319-81&amp;nbsp;</td><td></td></tr><tr><td>Brefeldin A-containing solution: GolgiPlug</td><td>BD Biosciences</td><td>555029</td><td></td></tr><tr><td>cDNA synthesis kit: SuperScript VILO(Reverse transcriptase) cDNA Synthesis Kit</td><td>Invitrogen</td><td>11754-250</td><td></td></tr><tr><td>Density centrifugation medium: Ficoll-Paque</td><td>GE healthcare</td><td>17-1440-03</td><td></td></tr><tr><td>DMSO 99.7%</td><td>Sigma Aldrich</td><td>D2650-5X5ML</td><td></td></tr><tr><td>FBS, heat inactivated</td><td>Invitrogen</td><td>10082-147</td><td></td></tr><tr><td>Fixable Viability Dye, eFluor 780&amp;nbsp;&amp;nbsp;</td><td>eBioscience</td><td>65-0865-14 or 65-0865-18&amp;nbsp;</td><td></td></tr><tr><td>Foxp3 Staining Buffer Set</td><td>eBioscience</td><td>00-5523-00</td><td>Caution: contains paraformaldehyde. Can be also bought in combined kit with antibody; 77-5774-40 Anti-Human Foxp3 Staining Set APC Clone: 236A/E7 Set</td></tr><tr><td>GlutaMAX (200 mM L-alanyl-L-glutamine)</td><td>Invitrogen</td><td>35050-061</td><td></td></tr><tr><td>human naive CD4 T cell isolation kit II</td><td>Miltenyi Biotec</td><td>130-094-131</td><td></td></tr><tr><td>Human serum albumin 50 g/L</td><td>Baxter</td><td>1501057</td><td></td></tr><tr><td>Ionomycin from Streptomyces conglobatus &gt;98%</td><td>Sigma Aldrich</td><td>I9657-1MG</td><td></td></tr><tr><td>MACS LS-columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>mouse IgG1 K Isotype Control APC Clone: P3.6.2.8.1</td><td>eBioscience</td><td>17-4714-42</td><td></td></tr><tr><td>mouse IgG1 K Isotype Control FITC 50 &amp;#956;g</td><td>eBioscience</td><td>11-4714-81&amp;nbsp;</td><td></td></tr><tr><td>mouse IgG2a isotype control, Brilliant violet 421, clone MOPC-173</td><td>BD Biosciences</td><td>563464</td><td></td></tr><tr><td>Pasteur pipet plastic, individually packed</td><td>Sarstedt</td><td>86.1172.001&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>PMA Phorbol 12-myristate 13-acetate</td><td>Sigma Aldrich</td><td>P1585-1MG&amp;nbsp;</td><td></td></tr><tr><td>Rapamycin</td><td>EMD (Merck)</td><td>553210-100UG</td><td></td></tr><tr><td>Recombinant Human IL-2, CF</td><td>R&amp;amp;D</td><td>202-IL-050/CF</td><td></td></tr><tr><td>Recombinant Human TGF-beta 1, CF</td><td>RnD</td><td>240-B-010/CF</td><td></td></tr><tr><td>RNA isolation kit: RNAqueous-Micro Kit</td><td>Ambion</td><td>AM1931&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>RPMI 1640 Medium&amp;nbsp;</td><td>Invitrogen</td><td>72400-054&amp;nbsp;</td><td></td></tr><tr><td>Sodium butyrate</td><td>Sigma Aldrich</td><td>B5887-250MG&amp;nbsp;</td><td></td></tr><tr><td>T cell culture medium: X-Vivo 15 medium, with gentamicin+phenolred</td><td>Lonza</td><td>04-418Q</td><td></td></tr><tr><td>TaqMan Gene Expression Assay, FOXP3 (Best Coverage)&amp;nbsp;</td><td>Applied Biosystems</td><td>4331182; assay ID: Hs01085834_m1</td><td>Caution: contains paraformaldehyde</td></tr><tr><td>TaqMan Gene Expression Assay, RPL13A (Best Coverage)</td><td>Applied Biosystems</td><td>4351370; assay ID: Hs04194366_g1&amp;nbsp;&amp;nbsp;</td><td>Caution: contains paraformaldehyde</td></tr><tr><td>TaqMan Gene Expression Master mix</td><td>Applied Biosystems</td><td>4369514</td><td></td></tr></tbody></table>

in vitro differentiation of human cd4+foxp3+ induced regulatory t cells (itregs) from na&#239;ve cd4+ t cells using a tgf-&#946;-containing protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

도 1은 실험 장치의 구조를 도시한다. 그림 2는 자기 절연 순진한 CD4 + T 세포와 nTregs에 대한 대표적인 순도 제어 염색을 보여줍니다. F의 igure 3a는 게이팅 전략 세포 계측법 흐름을 보여주고 그림 3b는 대표 Foxp3를하고 CD25가 표시 iTreg 또는 제어 조건 하에서 문화의 날 (...

Watch this Scientific Journal Video about In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells iTregs from NaÃ¯ve CD4+ T Cells Using a TGF-Î²-containing Protocol at JoVE.com

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

이 프로토콜은 체외 나이브 CD4 + T 세포로부터 재생 가능한 생성 유도 인간 T 조절 세포 (iTregs)의 표현형을 설명한다. Foxp3의 유도를위한 다른 프로토콜은 각각의 프로토콜로 얻은 특정 iTreg의 표현형 연구를 허용합니다.

TGF-β 함유 프로토콜을 사용하여 나이브 CD4 + T 세포에서 인간의 CD4 + Foxp3의 + 유도 규제 T 세포 (iTregs)의 체외 분화

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

in-vitro-differentiation-human-cd4-foxp3-induced-regulatory-t-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

20.1K 조회수.  Karolinska Institutet, Karolinska University Hospital, & Science for Life Laboratory.  이 프로토콜은 체외 나이브 CD4 + T 세포로부터 재생 가능한 생성 유도 인간 T 조절 세포 (iTregs)의 표현형을 설명한다. Foxp3의 유도를위한 다른 프로토콜은 각각의 프로토콜로 얻은 특정 iTreg의 표현형 연구를 허용합니다. 

비디오: In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Naïve CD4+ T Cells Using a TGF-β-containing Protocol

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

사람의 체외에서</em면역 불균형의 초기 국가의 인정을위한 심사 도구를> 억제

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

연구

면역학 및 감염병학

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

기본 인적 나이브 및 메모리 T의 세포로부터 유도된 규제 T 세포의 생성

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

T Lymphocytes쪽으로 유도 Pluripotent 줄기 세포의 분화 감독

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

새로운 도구는 HIV-1에 감염된 개인으로부터 규제 T 세포를 확장하려면

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

나이브 CD4의 아데노 바이러스 형질은 Treg의 분화를 연구하는 세포를 T

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

분리 및 나이브 CD4 T 림프구의 TH17 분화

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

마우스 나이브 CD4<sup&gt; +</sup&gt; T 세포의 분리 및<em&gt; 시험관</em&gt; T 세포 하위 집합으로 차별화

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

자가 면역 대해 줄기 세포 유래의 항원 특이 적 조절 T 세포의 발달

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Generation of Human Regulatory T Cell Clones

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based on the identification of Treg through several activation-dependent cellular surface markers with varying expression levels in different physiologic and pathologic conditions. Populations isolated as &#8220;Treg&#8221; therefore often contain considerable numbers of non-Treg effector cells (i.e., Teff) which hamper the precise phenotypic and functional characterization of these cells, their genomic and proteomic characterization, their reliable enumeration in different states of health and disease, as well as their isolation and expansion for therapeutic purposes. The latter, in particular, remains a major hurdle, as the inadvertent expansion of effector cells homing in Treg-relevant cellular compartments (e.g., CD4+CD25+ T cells) may render Treg-based immunotherapy ineffective, or even harmful. This work presents a method that circumvents the problems associated with population-based isolation and expansion of Treg and shows that the generation of Treg candidate clones with the subsequent selection, culture, and expansion of only carefully vetted, monoclonal cells, enables the generation of an ultrapure Treg cell product that can be kept in culture for many months, enabling downstream investigation of these cells, including for possible therapeutic applications.

This protocol describes the cloning and expansion of human regulatory T cells for the generation of ultra-high purity viable human Treg with stable demethylation at the Treg-specific demethylated region (TSDR) and Treg-specific phenotypic features.

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based ...

<meta charset="utf8"></head><body>유세포 분석을 사용한 Naive CD4+ T 세포에서 Th17 세포 분화

In Vitro Differentiation of Naive CD4+ T Cells into Pathogenic Th17 Cells in Mouse

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have been linked to a wide range of diseases in recent times, including multiple sclerosis (MS), rheumatoid arthritis, acute respiratory distress syndrome (ARDS), sepsis, and other autoimmune disorders. However, the currently known in vitro differentiation protocols have difficulty achieving high purity of pathogenic Th17 cells, with the induction efficiency often below 50%, which is a key challenge in in vitro experiments. In this protocol, we propose an enhanced in vitro culture and differentiation protocol for pathogenic Th17 cells, which is used to directly differentiate naive CD4+ T cells isolated from mouse spleens into pathogenic Th17 cells. This protocol provides detailed instructions on splenocyte isolation, purification of naive CD4+ T cells, and differentiation of pathogenic Th17 cells. Through this protocol, we can achieve a differentiation purity of approximately 90% for pathogenic Th17 cells, which meets the basic needs of many cellular experiments.

<meta charset="utf8"></head><body>저자 스포트라이트: 사이토카인 농도 조절을 사용하여 Th17 세포의 고순도 In Vitro 분화 달성

This protocol describes the differentiation of na&#239;ve CD4+ T cells into pathogenic Th17 cells in vitro. Specifically, when combined with a multi-parameter flow cytometry-based approach, 90% purity of pathogenic Th17 cells can be obtained from na&#239;ve CD4+ T cells using this differentiation method.

마우스에서 Naive CD4+ T 세포를 병원성 Th17 세포로 체외에서 분화

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have ...

TGF-β 함유 프로토콜을 사용하여 나이브 CD4 ⁺ T 세포에서 인간의 CD4 ⁺ Foxp3의 ⁺ 유도 규제 T 세포 (iTregs)의 체외 분화

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