The authors would like to acknowledge Dr. Laura Johnson and Dr. Richard Morgan for providing the CAR retroviral construct. The authors also thank Giao Ngyuen for her assistance with dosimetry for whole brain irradiation. This work was supported by an NIH NCI grant 1R01CA177476-01.

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The authors have no conflicts of interests to declare.

ציר זמן הטיפול המתואר כאן נועד מודל הסטנדרטי של טיפול קליני ולמנף את השפעתו לטיפול מאמצת CAR. מינוני CAR T תא, משטרי TMZ, וממשל הקרנות יכולים להיות שונה כדי לשפר את הפעילות בvivo T תא, lymphodepletion, והרג גידול. ניתן להגדיל משטרי TMZ להניב myeloablation מארח והתרחבות מוגברת של תאים הוע?...

גליובלסטומה (GBM) היא הגידול במוח הנפוץ ביותר הראשוני הממאיר והוא תמיד קטלני. כריתה כירורגית בשילוב עם כימותרפיה סטנדרטית של טיפול והקרנות שאינם ספציפי לא מצליחה לחסל לחלוטין תאים ממאירים, וכתוצאה מכך הפרוגנוזה עגומה של פחות מ -15 חודשים בחולים עם מחלה זו 1. בניגוד לכך, טיפול חיסוני מציע גישה מדויקת לאופן ספציפי מיקוד תאים סרטניים, ובכך יש לו הפוטנציאל לשמש פלטפורמת טיפול יעילה ביותר עם ​​סיכון מופחת של רעילות בטחונות 2-4. תאי T מהונדסים vivo לשעבר להביע קולטנים אנטיגן chimeric (מכוניות) מציעים אסטרטגיה רב-תכליתית לטיפול חיסוני גידול. מכוניות שנוצרו על ידי פיוזינג האזור משתנה תאי של נוגדן עם מולקולת תא T תאית אחד או יותר איתות (s), במקום histocompatibility הגדול באורך מלא מורכב (MHC) -restricted קולט תא T 5. מצב זה של פרסים והכרה אנטיגן כמו-נוגדןבמאפשר לתאי T אנטיגן הספציפי לתגובתי לזהות ולהגיב לאנטיגנים סרטניים בהעדר MHC ויכולים להיות מותאמים לרפרטואר אנטיגן כמעט אינסופי. תאי T CAR המהונדסים נגד מגוון רחב של אנטיגנים סרטניים הראו יעילות קלינית ופרה הבטחה מצטיינת במרפאת 6-9. באופן ספציפי, בהקשר של GBM, גרסת CAR פלטפורמת תא T מיקוד צמיחת אפידרמיס קולטן גורם III (EGFRvIII), מוטציה גידול ספציפי הביעה על פני תא 10, הוצג להארכת הישרדות בעכברי נושאי גליומה 11. למרות צדדיות שלהם, לעומת זאת, התועלת הקלינית של טיפול המאמץ CAR לא התממשה במלואו, נובע בחלקו הדיכוי החיסוני גידולים הקשורים והעלמת חיסונית 12-16, כמו גם אתגרים בהקמה ואחזקת תאי T אנטיגן הספציפי in vivo. מינוף הטיפול סטנדרטי (SOC) עם טיפול חיסוני עלולה להתגבר כמה מאלה lחיקויים, וכתוצאה מכך היעילות משופרת בשתי ההגדרה פרה-קלינית וקלינית. SOC להודעה כריתה-GBM מורכב מtemozolomide במינון גבוה (TMZ), סוכן אלקילציה DNA 17, והקרנת המוח כולו (WBI) 1. טיפולים אלה בחזקתו יתמזגו עם חיסוני גידול באמצעות גברת ביטוי של גידול ביטוי MHC 18-20 ושפיכת אנטיגנים על ידי תאים סרטניים מתים 17,19,21,22. ואכן, התוספת של TMZ 20,23 או WBI 18,24 מוביל ליעילות אנטי-סרטנית משופרת של טיפולים מבוססי חיסון בהגדרה פרה-קלינית. יתר על כן, כמו רבים chemotherapeutics ציטוטוקסיות שאינו ספציפי, TMZ ידוע כגורם 25,26 lymphopenia מערכתי, שניתן למנף כאמצעי למארח-אוויר לפלטפורמות טיפול מאמצים 27-29. lymphodepletion תיווך TMZ הוכח כדי לשפר את התדירות ואת התפקוד של תאי T אנטיגן ספציפי, שמוביל ליעילות מוגברת של adopפלטפורמת טיפול מופרזת נגד גידולים תוך-גולגולתי 30. בהקשר של טיפול CAR, lymphodepletion משמש כאמצעי למארח-אוויר על ידי שני צמצום מספר מדכא אנדוגני תאי T 31, וגרימת תרבות הומיאוסטטית 32 באמצעות הפחתת תחרות לציטוקינים 33, וכך לשפר את הפעילות אנטי-סרטני 11,34. בהתחשב ביחסים סינרגטיים בין פלטפורמות SOC ואימונותרפיה GBM, הערכת טיפולים מאמצים רומן ופלטפורמות חיסון בהקשר של SOC היא קריטית להסקת מסקנות משמעותיות לגבי יעילות. בפרוטוקול זה, אנו מתארים שיטה לדור והעירוי לוריד של תאי T CAR העכבריים EGFRvIII הספציפי לצד TMZ וWBI בעכברי נושאי גידולים תוך-גולגולתי EGFRvIII-חיובי (ראה תרשים 1 לציר זמן טיפול). בקצרה, תאי T CAR נעשים לשעבר vivo על ידי התמרה retroviral. כליה עוברית אנושית (HEK) 293T תאים transfected באמצעות מורכב DNA / שומנים בדם (המכיל את וקטור CAR ופלסמידים PCL-Eco) לייצר וירוס, אשר לאחר מכן נעשה שימוש כדי transduce splenocytes העכברי הופעל שנקצרים ותרבותי במקביל. במהלך דור CAR, מארחים עכבריים נושאות גידולים תוך-גולגולתי EGFRvIII חיובית מנוהלים הקרנה מופרד המוח כולו רנטגן וטיפול TMZ מערכתי במינונים דומים לSOC הקליני. לאחר מכן תאי T CAR מועברים דרך הווריד למארחי lymphodepleted. ההליך הבא מתואר בשבעה שלבים נפרדים: (1) מנהל של temozolomide לעכברים, (2) הקרנת המוח כולו של עכברים נושאי גידול, נושאי גידול (3) Transfection, (4) כריתת טחול ותא T הכנה, (5 ) התמרה, (6) תרבות תא T CAR וקציר, ו( 7) ממשל תא T CAR לעכברי נושאי גידול. שלבים אלה מורכבים מכמה שלבים שמשתרעים על פני 6-7 ימים ומבוצעים במקביל.

<table border="0" cellpadding="0" cellspacing="0" width="1130">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Name of Material</td>
			<td style="width:172px;">Company</td>
			<td style="width:125px;">Catalog Number</td>
			<td style="width:449px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCL-Eco Retrovirus Packaging Vector</td>
			<td>Imgenex</td>
			<td>10045P</td>
			<td>Helper vector for generating CAR retrovirus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Concanavalin A</td>
			<td>Sigma Aldrich</td>
			<td>C2010</td>
			<td>Non-specific mitogen to induce T cell proliferation and viral transduction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Retronectin</td>
			<td>ClonTech/Takara</td>
			<td>T100B</td>
			<td>Facilitates retroviral transduction of T cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine 2000</td>
			<td>Life Technologies</td>
			<td>11668-019</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, high glucose, pyruvate</td>
			<td>Life technologies</td>
			<td>11995-065</td>
			<td>HEK293 culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td>T cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEM I Reduced Serum Medium</td>
			<td>Life technologies</td>
			<td>11058-021</td>
			<td>Transfection media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 mM L-Glutamine&#160;</td>
			<td>Life technologies</td>
			<td>25030-081</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mM Sodium Pyruvate</td>
			<td>Life technologies</td>
			<td>11360-070</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100X MEM Non-Essential Amino Acids Solution&#160;</td>
			<td>Life technologies</td>
			<td>11140-050</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">55 mM 2-Mercaptoethanol&#160;</td>
			<td>Life technologies</td>
			<td>21985-023</td>
			<td>Reducing agent to remove free radicals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life technologies</td>
			<td>15140-122</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamicin (50 mg/mL)&#160;</td>
			<td>Life technologies</td>
			<td>15750-060</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GemCell U.S. Origin Fetal Bovine Serum</td>
			<td>Gemini Bio Products</td>
			<td>100-500</td>
			<td>Provides growth factors and nutrients for in vitro cell growth&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin (BSA), Fraction V&#8212;Standard&#160;Grade&#160;</td>
			<td>Gemini Bio Products</td>
			<td>700-100P</td>
			<td>Blocks non-specific binding of retrovirus to retronectin-coated plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pharm Lyse (10X concentrate)&#160;</td>
			<td>BD Biosciences</td>
			<td>555899</td>
			<td>Lyses red blood cells during splenocyte processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70 &#181;m Sterile Cell Strainers</td>
			<td>Corning</td>
			<td>352350</td>
			<td>Filters away large tissue particles during splenocyte processing&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mm BioCoat Culture Dishes with Poly-D-Lysine&#160;</td>
			<td>Corning</td>
			<td>356469</td>
			<td>Promotes HEK293 cell adhesion to maximize proliferation after transfection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Temozolomide</td>
			<td>Best Pharmatech</td>
			<td>N/A</td>
			<td>Lyophilized powder prepared on the day of administration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl Sulfoxide</td>
			<td>Sigma Life Sciences</td>
			<td>D2650</td>
			<td>Necessary for complete dissolution of temozolomide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline</td>
			<td>Hospira</td>
			<td>IM 0132 (5/04)</td>
			<td>Solvent for temozolomide and ketamine/xylazine&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketathesia HCl</td>
			<td>Henry Schein Animal Health</td>
			<td>11695-0701-1</td>
			<td>Ketamine solution&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AnaSed</td>
			<td>Lloyd Inc</td>
			<td>N/A</td>
			<td>Xylazine sterile solution 100 mg/mL&#160;</td>
		</tr>
	</tbody>
</table>

generation of car t cells for adoptive therapy in the context of glioblastoma standard of care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

תאי T CAR מופקים על ידי התמרה עם וקטור retroviral EGFRvIII CAR 11. וקטור זה, MSGV1, פותח מוקטור SFGtcLuc_ITE4 35, המכיל את הווירוס בתאי גזע העכבריים (MSCV) חוזר מסוף ארוך, אזור איסור פרסום ואתר שחבור מעטפה המורחב (תורם אחוי, SD, ואחוי acceptor, sa), וויראלי אות אריזה (ψ). CAR EGFRvIII מכיל בר האדם אנט?...

Watch this Scientific Journal Video about Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care at JoVE.com

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

דור של תאי T CAR למאמצת תרפיה בהקשר של גליובלסטומה טיפול סטנדרטי

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

generation-car-t-cells-for-adoptive-therapy-context-glioblastoma

Research

JoVE Journal

Immunology and Infection

21.2K Views.  Duke University. The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care. 

Video: Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

התמרה retroviral של T-cell קולטנים עכבר מתאי T

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

דור Multivirus ספציפיים תאים T כדי למנוע / לטפל זיהומים נגיפיים לאחר השתלת allogeneic תא גזע hematopoietic

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

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&amp;#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.

דור של מושרה תאים T רגולטוריים של האדם תאים ראשוניים טי נאיביות וזיכרון

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

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

שינוי מערכת Transposon piggyBac של תאי T אנושיים ראשוניים

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

פפטיד: העשרת tetramer מבוסס MHC של תאי T epitope ספציפיים

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

אינדוקציה מואצת סוכרת סוג 1 בעכברים על ידי העברת המאמצת של CD4 + T תאי Diabetogenic

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

אינדוקציה של Murine דלקת מעיים על ידי העברת המאמצת של מפעיל CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; גבוה</sup&gt; תאי T לעכברי immunodeficient

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

העשרת תא אוטומטית של ציטומגלווירוס ספציפי תאי T ליישומים קליניים באמצעות מערכת ציטוקין ללכוד

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

העברת סטיאוטטי המאמצת של תאים חיסוניים ציטוטוקסיות במודלים מאתר של האדם Orthotopic גליובלסטומה Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

Production of Human CRISPR-Engineered CAR-T Cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.