The authors wish to thank Dr. Boris Reizis from the Department of Microbiology and Immunology at Columbia University for helpful discussions. We also wish to thank Dr. Margaret Bynoe, Dr. Rodman Getchell and Deequa Mahamed from the Department of Microbiology and Immunology at the College of Veterinary Medicine, Cornell University for technical assistance with flow cytometry and Lu Huang for assistance with video editing. The authors also wish to thank the members of Duhamel and Weiss laboratories for their critical review of the manuscript and video production, and Stephanie Yazinski from the Weiss laboratory for breeding and genotyping p53-/- mice. Experiments on animals were performed in accordance with the guidelines and regulations set forth by Cornell University Institutional Animal Care and Use Committee. This research was supported in part by funds provided by the US Department of Agriculture, Cooperative State Research, Education, and Extension Service, Animal Health and Disease Research Program (to G.E.D. and R.S.W.) and NIH grants R03 HD058220 and R01CA108773 (to R.S.W). The video was produced using in-house facilities by the personnel from the Duhamel &amp; Weiss laboratories.

1. Dissection of Tumor
<ol>
	<li>For tumor dissection, a clean paper towel or disposable surgical drape is placed on the dissection pad and sprayed with 70% ethanol.</li>
	<li>Mice for this protocol are routinely euthanized with CO2.Place the euthanized mouse on the dissection pad and spray both sides of the animal with ethanol. The mouse is held in place onto the dissection pad by using four or more push pins inserted through the legs and sprayed thoroughly with 70% ethanol.</li>
	<li>The skin over the midline is cut from the pubis to the base of the head by using sterile scissors, while avoiding the underlying muscles and penetrating into the body cavities.</li>
	<li>Open the abdominal cavity with a new pair of sterile scissors and forceps being careful to not damage the internal organs.</li>
	<li>Open the thoracic cavity by dissecting and deflecting the rib cage to reveal the tumor, without damaging blood vessels.</li>
	<li>Separate the tumor from other organs, and transfer a piece approximately 40-50% of the total tumor (approximately up to 1.5 X 0.8 X 0.8 cm) into cold PBS kept on ice.</li>
	<li>The remaining portion of the tumor can be saved for other assays and a complete necropsy can be performed at this time.</li>
</ol>
2. Culturing Lymphoma Cells
<ol>
	<li>Spray the tube containing the dissected tumor with 70% ethanol and transfer to a biosafety cabinet.</li>
	<li>Add 10 mL of culture medium to a sterile 150 mm2 Petri dish.</li>
	<li>Transfer the piece of tumor into the Petri dish by gentle suction with a pipette.</li>
	<li>Bend two sterile 18 gauge needle facing the beveled side out.</li>
	<li>Dissociate the tumor using the bent needles.</li>
	<li>Swirl the dish, tap gently and place at an angle to collect the cell suspension to a side, while avoiding large pieces of tumor.</li>
	<li>Aspirate the cell suspension and transfer into a 50 mL conical tube while avoiding large pieces of tumor.</li>
	<li>Wash the plate with 10 mL of medium and transfer into the same tube while avoiding large pieces of tumor.</li>
	<li>Mix the cell suspension composed of lymphocytes and stromal cells gently and transfer 5 mL into a T-25 tissue culture flask and 10 mL into a T-75 tissue culture flask. Gently swirl the flasks.</li>
	<li>Use the remaining cell suspension to make 2, 4, 10,100 and 1000 fold dilutions in 2.5 mL final volume (culturing cells at different densities using several types of culture vessels maximizes the likelihood of establishing a T-cell line).</li>
	<li>Plate 2.0 mL of each dilution into individual wells of a 6-well plate. Gently swirl the plate.</li>
	<li>Incubate the cells for 48 h in humidified CO2 atmosphere in air.</li>
</ol>
3. Feeding Lymphoma Cells
<ol>
	<li>After 48 h of incubation, add 5 mL of medium to the T-25 flask and 10 mL of medium to the T-75 flask along the wall with minimal disturbance of the cells.</li>
	<li>Add 2 mL of medium to each well of the 6-well plate along the wall with minimal disturbance of the cells.</li>
	<li>Incubate the cells for an additional 72 h.</li>
</ol>
4. Passaging Lymphoma Cells
<ol>
<li>Initial passage
<ol>
<li>At the end of the fifth day of incubation, the cells are passaged at a 1:1 ratio, while retaining the original flasks and plates (maintaining the cell density at a concentration of approximately 3x106/ mL during the first passages promotes successful establishment of a continuous cell line).
<ol class="lalpha">
<li>For the T-25 flask, transfer 5 mL of cell suspension into a new flask containing 5 mL of medium. Add 5 mL of medium into the original flask.</li>
<li>For the T-75 flask, transfer 10 mL of cell suspension into a new flask containing 10 mL of medium. Add 10 mL of medium into the original flask.</li>
<li>For the 6-well plate, transfer 2 mL of cell suspension from each well into corresponding wells of a new plate containing 2 mL of medium in each well. Add 2 mL of medium into each well of the original wells.</li>
</ol>
</li>
<li>Incubate all flasks and plates for an additional 72 h. In our experience most cell lines are established from these passage one cultures (the presence of loosely adherent suspension cell aggregates over the attached stromal cells is an early indication of an established cell line).</li>
<li>For the cultures with fewer or no loosely adherent suspension cell aggregates, feed the flasks or plates by carefully replacing approximately 50% of the medium every 72 h. To avoid removing cells from the original culture, culture vessels are left standing and undisturbed for at least 5 min to let the cells sediment at the bottom by gravity before gentle aspiration of the supernatant medium.</li>
</ol>
</li>
<li>Maintaining established cell lines
<ol>
<li>Established cell lines are passaged into new flasks and plates. For T-25 and T-75 flasks, add 5 mL of the original or passage one suspension into 5 mL of medium. For 6-well plates, add 3 mL of the original or passage one cell suspension into 7 mL of medium in a T-25 flask. Feed and retain the original or passage one culture by replacing the volume removed with fresh culture medium.</li>
<li>Passage established cell lines into new T-25 flasks. The cell lines have a doubling time of 18-24 h. The cultures are routinely maintained at a density of 1-3x106/ mL and passaged every 3 days. Add the required volume of previous passage culture to new medium to make up to 10 mL. Feed and retain the original culture.</li>
<li>After a few passages, the non-adherent cells will self sustain without the adherent stromal cells and a smaller number of adherent cells are carried over with each passage. Beyond this point, the cultures can be maintained in non-tissue culture treated T-25 flasks.</li>
</ol>
</li>
</ol>
5. Freezing and Recovering Lymphoma Cell Lines
<ol>
	<li>The cell lines are frozen in freshly prepared RPMI 1640 medium containing 20% FBS and 10% DMSO.</li>
	<li>Cells from a 48 h old culture in T-25 flask (approximately 1.5x106 cells/ mL) are centrifuged at 1500 rpm for 5 min at room temperature. After discarding the supernatant, the cells are re-suspended in 2 mL of cold freezing medium. Two 1.0 mL volumes of cell suspension from each flask are placed in freezing vials, immediately frozen at -80&deg;C, and transferred to liquid nitrogen the following day.</li>
	<li>Cells are recovered from liquid nitrogen storage by quick thawing, and resuspending the contents in 10 mL of fresh culture medium. Alternatively, freshly thawed cells are washed once in warm culture medium to remove the DMSO before resuspending in 10 mL of fresh culture medium. The cells are cultured for at least 72 h before subsequent passage.</li>
</ol>
6. Representative Results
An Atm-/- T-cell line developed using this protocol and designated DWJ-Atm-1 was characterized by staining with anti-CD3-FITC, anti-CD4-APC and anti-CD8-PE and flow cytometry analysis (Figure1). More than 90% of the T-cells were CD3, CD4 and CD8 triple-positive.
<img src="/files/ftp_upload/2598/2598fig1.jpg" alt="Figure 1" /> 
Figure 1. Characterization of a T-cell line developed using this protocol.
The cell surface markers of an established Atm-/- cell line designated DWJ-Atm-1 were characterized by staining with anti-CD3-FITC, anti-CD4-APC, anti-CD8-PE and flow cytometry analysis. Briefly, 1X106 fresh cells were washed once in cold PBS (1500 rpm/5 min), and incubated at 4&deg;C for 10 min with 200 &mu;L of antibody cocktail (each antibody at 1:200 dilution in PBS with 1% BSA. Cells can be examined immediately while fresh or fixed for later analysis. Fixed cells are prepared by adding neutral buffered formalin at a 4% (vol/vol) final concentration and incubated at 4&deg;C for 30 min, harvested by centrifugation (1500 rpm/5 min) and resuspended in 200 &mu;L of PBS with 1% BSA. A total of 1 X 104 cells were analyzed by using a BD FACS Calibur analyzer and CellQuest software (BD Biosciences, San Jose, CA).

<ol>
	<li>Barlow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atm-deficient+mice:+a+paradigm+of+ataxia+telangiectasia.">Atm-deficient mice: a paradigm of ataxia telangiectasia.</a> Cell. 86, 159-171 (1996).</li><li>Elson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleiotropic+defects+in+ataxia-telangiectasia+protein-deficient+mice.">Pleiotropic defects in ataxia-telangiectasia protein-deficient mice.</a> Proc Natl Acad Sci U S A. 93, 13084-13089 (1996).</li><li>Kuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Atm-/-+thymic+lymphoma+cell+proliferation+in+vitro+and+in+vivo+by+dexamethasone.">Control of Atm-/- thymic lymphoma cell proliferation in vitro and in vivo by dexamethasone.</a> Cancer Chemother Pharmacol. 55, 203-212 (2005).</li><li>Browne, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+with+a+catalytic+antioxidant+corrects+the+neurobehavioral+defect+in+ataxia-telangiectasia+mice.">Treatment with a catalytic antioxidant corrects the neurobehavioral defect in ataxia-telangiectasia mice.</a> Free Radic Biol Med. 36, 938-942 (2004).</li><li>Jacks, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+spectrum+analysis+in+p53-mutant+mice.">Tumor spectrum analysis in p53-mutant mice.</a> Curr Biol. 4, 1-7 (1994).</li><li>Chervinsky, D. S., Lam, D. H., Zhao, X. F., Melman, M. P., Aplan, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+T+cell+leukemia+cell+lines+established+from+SCL/LMO1+double+transgenic+mice.">Development and characterization of T cell leukemia cell lines established from SCL/LMO1 double transgenic mice.</a> Leukemia. 15, 141-147 (2001).</li><li>Sharma, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch1+contributes+to+mouse+T-cell+leukemia+by+directly+inducing+the+expression+of+c-myc.">Notch1 contributes to mouse T-cell leukemia by directly inducing the expression of c-myc.</a> Mol Cell Biol. 26, 8022-8031 (2006).</li><li>Maser, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomally+unstable+mouse+tumours+have+genomic+alterations+similar+to+diverse+human+cancers.">Chromosomally unstable mouse tumours have genomic alterations similar to diverse human cancers.</a> Nature. 447, 966-971 (2007).</li></ol>

No conflicts of interest declared.

In this protocol, we provide detailed procedures to establish murine T-cell lines from two different mouse genotypes; Atm-/- [FVB/N-Atmtm1Led/J] and p53-/- [129/S6-Trp53tm1Tyj/J]. By using this protocol, a total of 6 (out of 7 attempts) Atm-/- and three (out of 5 attempts) p53-/- murine T-cell lines have been established in our laboratory. Representative data demonstrating that cell line DWJ-Atm-1, an<e...

<ul>
<li>Ice bucket</li>
<li>70% ethanol</li>
<li>Dissection pad consisting of foam block covered with aluminum foil and pins</li>
<li>Two sets of small sterile scissors and forceps</li>
<li>Sterile 150 mm2 Petri dish (Thermo Fisher Scientific, Waltham, MA; cat. #08-757-13)</li>
<li>Sterile 18 gauge needles</li>
<li>50 mL conical tubes (Corning Inc., Corning NY; cat # 430829)</li>
<li>10% buffered formalin</li>
<li>Tissue culture flasks 
<ul>
<li>T-25 (TPP, Trasadingen, Switzerland; cat # 90025)</li>
<li>T-75 (TPP, Trasadingen, Switzerland; cat # 90075)</li>
</ul>
</li>
<li>6-well plates (Corning Inc., Corning NY; cat # 3506)</li>
<li>Sterile phosphate buffered saline pH 7.2 (PBS)</li>
<li>Culture medium 
<ul>
<li>RPMI 1640 with 25 mM HEPES, 200 mM L-glutamine (Lonza, Walkersville, MD; cat#12-115F) supplemented with the following:
<ul>
<li>10% heat inactivated (56&deg;C, 30 min) fetal bovine serum (FBS; Hyclone, Logan, UT; cat# SH30088.03)</li>
<li>1% penicillin and streptomycin (Mediatech, Manassas,VA; cat# 30-002-CI)</li>
<li>1% nonessential amino acids (Mediatech; cat#25-025-CI)</li>
<li>55 mM 2&beta;-mercaptoethanol (Sigma, St Louis, MO; cat#M752)</li>
</ul>
</li></ul>
</li>
<li>Dimethyl sulfoxide (DMSO; Calbiochem, La Jolla, CA; cat # 317275)</li>
<li>Antibodies (eBioScience, San Diego, CA)
<ul>
<li>anti-CD3-FITC (cat. #11-0031-85)</li>
<li>anti-CD4-APC (cat. #17-0041-83)</li>
<li>anti-CD8-PE (cat.#12-081-85)</li>
</ul>
</li>
<li>Bovine serum albumin (BSA; Life Technologies, Grand Island, NY; cat #. 11018-017)</li>
<li>Concanavalin A (Sigma cat# C5275)</li>
<li>1.8 mL freezing vials (Nunc, Roskilde, Denmark; cat# 368632)</li>
</ul>

derivation of thymic lymphoma t-cell lines from atm-/- and p53-/- mice

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm-/- and p53-/- consistently develop thymic lymphoma early in life 1,2, and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols 1,3. Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain 1,4. We have successfully established several T-cell lines using this protocol and inbred strains ofAtm-/- [FVB/N-Atmtm1Led/J] 2 and p53-/- [129/S6-Trp53tm1Tyj/J] 5 mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year.

Watch this Scientific Journal Video about Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice at JoVE.com

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

In this video we demonstrate a protocol to establish mouse thymic lymphoma cell lines. By following this protocol, we have successfully established several T-cell lines from Atm-/- and p53-/- mice with thymic lymphoma.

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

derivation-of-thymic-lymphoma-t-cell-lines-from-atm-and-p53-mice

Research

JoVE Journal

Immunology and Infection

12.2K 次观看.  Cornell University. 在这个视频中，我们展示了一个协议来建立小鼠胸腺淋巴瘤细胞株。通过这个协议，我们已经成功地建立了几个从ATM / T细胞线 - 和p53 - / - 小鼠胸腺淋巴瘤。

视频: Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

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&amp;#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.

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

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to proteins of interest. Identification of gene functions in cell line models is important for gene discovery and exploration of cell-intrinsic mechanisms. However, genetic manipulations of immune cells such as T cells and macrophage cell lines using CRISPR/Cas9-mediated knock-in are difficult because of the low transfection efficiency of these cells, especially in a quiescent state. To modify genes in immune cells, drug-resistance selection and viral vectors are typically used to enrich for cells expressing the CRIPSR/Cas9 system, which inevitably results in undesirable intervention of the cells. In a previous study, we designed dual fluorescent reporters coupled to CRISPR/Cas9 that were transiently expressed after electroporation. This technical solution leads to rapid gene deletion in immune cells; however, gene knock-in in immune cells such as T cells and macrophages without the use of drug-resistance selection or viral vectors is even more challenging. In this article, we show that by using cell sorting to aid selection of cells transiently expressing CRISPR/Cas9 constructs targeting the Rosa26 locus in combination with a donor plasmid, gene knock-in can be achieved in both T cells and macrophages without drug-resistance enrichment. As an example, we show how to express human ACE2, a receptor of SARS-Cov-2, which is responsible for the current Covid-19 pandemic, in RAW264.7 macrophages by performing knock-in experiments. Such gene knock-in cells can be widely used for mechanistic studies.

This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to ...

Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines

Extracellular vesicles (EVs) are a heterogeneous population of membrane vesicles released by cells in vitro and in vivo. Their omnipresence and significant role as carriers of biological information make them intriguing study objects, requiring reliable and repetitive protocols for their isolation. However, realizing their full potential is difficult as there are still many technical obstacles related to their research (like proper acquisition). This study presents a protocol for the isolation of small EVs (according to the MISEV 2018 nomenclature) from the culture supernatant of tumor cell lines based on differential centrifugation. The protocol includes guidelines on how to avoid contamination with endotoxins during the isolation of EVs and how to properly evaluate them. Endotoxin contamination of EVs can significantly hinder subsequent experiments or even mask their true biological effects. On the other hand, the overlooked presence of endotoxins may lead to incorrect conclusions. This is of particular importance when referring to cells of the immune system, including monocytes, because monocytes constitute a population that is especially sensitive to endotoxin residues. Therefore, it is highly recommended to screen EVs for endotoxin contamination, especially when working with endotoxin-sensitive cells such as monocytes, macrophages, myeloid-derived suppressor cells, or dendritic cells.

The proposed protocol includes guidelines on how to avoid contamination with endotoxin during the isolation of extracellular vesicles from cell culture supernatants, and how to properly evaluate them.