Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Podsumowanie

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

Streszczenie

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the “hanging drop” method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3⁺ cells and gave rise to the canonical γδ T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3^-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Wprowadzenie

T lymphocytes, bearing the αβ or the γδ T cell receptor (TcR), differentiate in a specialized organ, the thymus. The fully developed thymus is organized into two distinct regions: the cortex, where thymic progenitors develop and where thymocytes that productively rearrange the TcR β and α chain genes are rescued from programmed death (a process known as positive selection); and the medulla, where selected thymocytes with too strong reactivity to self-ligands are deleted (negative selection)^1,2. The thymus originates from the endodermal layer of the third pharyngeal pouch that is later surrounded by mesenchymal cells³. It is colonized by hematopoietic progenitors starting at embryonic day E12 and, thereafter, continuous recruitment is required for normal T cell development⁴. Thymic immigrants evolve through successive developmental stages, orchestrated by a tightly regulated program, initiated and maintained by the activation of the Notch signaling pathway on thymocytes upon interaction with its ligand, delta like 4, expressed on thymic epithelial cells (TECs)⁵.

Thymocyte development starts at the so-called CD4^-CD8^- double negative (DN) stages. DN thymocytes can be further subdivided according to the expression of CD25 and CD44 into DN1 (CD25^-CD44⁺), DN2 (CD25⁺CD44⁺), DN3 (CD25⁺CD44^-) and DN4 (CD25^-CD44^-). CD24 (HSA) and CD117 (c-Kit) further subdivides the DN1 compartment into 5 subsets where DN1a and b correspond to early thymic progenitors (ETP). Thymocytes rearrange the TcR δ, β and α chains at the DN stage and undergo pre-TcR selection (DN3-DN4 stages). They further transit to the CD4⁺CD8⁺ double positive (DP) compartment where the TcR α chain rearranges prior to positive and negative selection. At this stage most thymocytes are eliminated and only a small percentage (3-5%) reach the CD4⁺ or CD8⁺ mature T cell compartment.

The lymphoid differentiation pathway progresses through the stages of HSCs that generate multipotent progenitors (MPP) and lymphoid-primed multipotent progenitors (LMPP) that lost the erythrocyte and megakaryocyte potential⁶. LMPP are phenotypically defined by the absence of differentiated blood cell markers (lineage negative, Lin^-), the expression of c-Kit (CD117), Sca-1 and Flt3/Flk2 (CD135) and the absence of detectable levels of the interleukin (IL)-7 receptor α chain (IL-7rα or CD127). LMPPs further differentiate into common lymphoid progenitors (CLP)⁷ that by that stage have lost the capacity to generate myeloid cells. CLP retain lymphocyte (B and T cell), NK cell, DC and innate lymphoid cell (ILC) potential, and differ from LMPP by the expression of CD127 and the absence of high levels of Sca-1.

Although the nature of the thymic settling progenitors (TSP) has been extensively debated⁸, it became recently clear that TSP change phenotype, differentiation potential and function, throughout development⁹. We performed in vitro and in vivo assays to characterize the TSP, isolated by FACS cell sorting from either E13 (first wave) or E18 (second wave). Fetal thymic organ cultures (FTOC) with irradiated thymic lobes colonized by equal numbers of E13 and E18 progenitors, bearing different allotypic markers, allowed following their progeny in a similar developmental environment and revealed cell intrinsic properties, different between both types of progenitors. Thymic lobes colonized by either E13 or E18 TSP allowed development without selection due to competition between both progenitors. In vivo transplantation of the colonized thymic lobes further showed that also the mature progeny of E13 and E18 TSP have different biologic properties in vivo. TSPs from the first wave rapidly generate T cells but give rise to low numbers of αβ and γδ T cells. Among the latter we detected Vγ5Vδ1 dendritic epithelial T cells (DETC), that have an invariant TcR, migrate to the epidermis where they exert a function in wound healing and are only produced during embryonic development¹⁰. In contrast, TSP from the second wave take longer time to generate high numbers of TcR⁺ T cells and are unable to generate DETC.

Protokół

Ethics statement: all experiments were performed according to the Pasteur Institute Ethic Charter, approved by the French Agriculture Ministry, and to the EU guidelines. A manipulator with training on small rodent surgery, certified by the French Ministry of Agriculture, performs all surgical interventions.
NOTE: See in annex Table 1 showing the 5-step plan procedure.

1. Selection of the Embryos

1. Use 36 C57BL/6 CD45.2 females, 12 females CD45.1, 12 C57BL/6 CD45.1 males and 12 C57BL/6 CD45.2 males. Crossing the females with either male genotype will produce embryos CD45.1/2 and CD45.1 used as a source of TSP or CD45.2 used as a source of recipient thymic lobes. House all males individually.
2. To obtain embryos for E18 TSP isolation, place 2 females in a cage with one male CD45.1 at 6 pm, 21 days before the grafting experiment. Check the presence of a vaginal plug the next day, before noon. Separate the plugged females.
3. To obtain E14 embryos to be colonized by TSP, repeat the procedure above (1.2), using males CD45.2, 17 days before the grafting experiment.
4. To obtain embryos to isolate E13 TSP, repeat the procedure above (1.2), using males C57BL/6 CD45.1, 16 days before the grafting experiment.

2. Dissection of the Embryos Under a Horizontal Laminar Flow Hood

NOTE: Two days before the grafting experiment.

1. Sacrifice the pregnant females by cervical dislocation or by progressive administration of CO₂ gas during at least 5 min, in a saturating closed chamber. Ensure death by definitive arrest of cardio-respiratory movements. Wet the abdomen with 70% ethanol.
2. Make a longitudinal incision in the skin at the midline abdomen and open it by pulling the skin apart. Open the peritoneum with forceps and scissors without touching the digestive tract. Pull out the bifid uterus and separate it from the vagina with the scissors.
3. Place the uterus in a 90 x 15 mm Petri dish containing 40-50 ml DPBS and cut transversally between each embryo’s decidua.
4. With the pair of fine tip scissors and a fine forceps remove the muscle membrane of the uterus, take out the embryos by cutting between the placenta and the yolk sac, and remove the amnios, which is retaining the embryos.
5. Place the embryos in a Petri dish 90 x 15 mm containing HBSS + 1% fetal calf serum (FCS). For embryos older than E15, remove the heads with a pair of scissors before any further dissection.

3. Isolation of the Thymus

1. Under the binocular magnifying lens, place the embryo, lying in supine position (ventral view), on a wet gauze bedding.
2. Insert one forceps longitudinally along the cartilage of the sternum, pinch and open the thoracic grid. With curved forceps, pull the thoracic wall aside to visualize the thymic lobes on each side of the trachea.
3. Carefully hold each lobe by pinching underneath with a fine forceps and place them in a Petri dish containing 1 ml HBSS + 1% FCS. Isolate the lobes and remove surrounding connective tissue with two 1 ml syringes + 26 G ⅜" needles, without damaging the capsule.
4. Wash the lobes in 3 ml of HBSS + 1% FCS to eliminate blood cells.
   NOTE: Before E15, thymic lobes are located on each side of the trachea and later fuse in the middle to constitute a bi-lobar organ.

4. Cell Suspensions

1. Tease apart the lobes under the binocular magnifying lens, with two 26 G needles mounted on 1 ml syringes, in HBSS + 1% FCS. Filter the cell suspension through a nylon mesh.

5. Staining with Fluorescent Antibodies and Cell Sorting

NOTE: All antibodies are previously titrated to obtain optimal definition of the populations (the titration can vary with the antibody batch).

1. Incubate the thymocytes (E13 or E18) for 15-30 min at 4 °C with a 100 μl of a cocktail of biotinylated antibodies to stain lineage positive cells (anti-CD19, anti-Gr1, anti-Ter119, anti-NK1.1, anti- CD11c, anti-CD3, anti-CD4, anti-CD8, anti-CD25).
2. To wash away excess of antibodies spin down the tubes with 4 ml HBSS + 1% FCS at 280 x g for 7 min, eliminate the supernatant and re-suspending the pellet in fresh HBSS + 1% FCS. Repeat the centrifugation.
3. Incubate the E18 biotin-labeled cells with streptavidin microbeads during 15 min at 4 °C and wash the cells twice in HBSS 1% FCS. Re-suspend the cells in 2 ml HBSS + 1% FCS. Most E13 thymocytes are lineage negative and therefore do not need MACS enrichment prior to cell sorting.
4. Pass the cells on the LS column, according to the manufacturer’s instructions. When all the cell suspension entered the column add 2 ml HBSS + 1% FCS. Recover the 4 ml of the column flow through and centrifuge the cells for 7 min at 280 x g.
5. Re-suspend the pellet of either depleted E18 or total E13 thymocytes in 50 μl of the TSP antibody mix (anti-CD117 APC, anti-CD135 PE, anti-CD127 PECy7, anti-CD24 FITC, anti-CD44 APCCy7 and streptavidin Pacific Blue) and incubate for 20 min at 4 °C in the dark.
6. Wash the cells with 4 ml HBSS + 1% FCS and re-suspend the cells in 1 ml HBSS + 1% FCS with propidium iodide.
7. Sort the TSPs Lin^-CD44⁺CD117⁺CD24^lowCD127⁺CD135⁺ cells in a FACS (with 4 lasers) onto a 1.5 ml tubes containing 200 μl complete medium + 20% FCS. Gate first the cells according to their size and granularity on the FSC and SSC channels. Eliminate dead cells (staining with propidium iodide), gate out doublets and score the fluorescence in exponential scales. Around 100 or 600 TSPs can be obtained from one E13 or one E18 thymus, respectively.

6. Colonization of E14 Thymic Lobes with Progenitors: Hanging Drop Technique

1. Irradiate (30 Grays = 30 Gy) E14 thymic lobes from CD45.2 embryos, 3 hr before culture.
   NOTE: E14 or E15 thymic lobes have been successfully used as recipient of T cell progenitors. Using recipient thymic lobes of later gestational days results in premature necrosis due to the bigger size of the organ while thymic lobes at earlier gestational days develop poorly, probably due to incomplete development of the epithelial cells.
2. Centrifuge sorted TSPs and re-suspend the cells in culture medium (OPTI-MEM complete medium with 10% FCS, penicillin (50 units/ml), streptomycin (50 µg/ml) and 2β-mercaptoethanol (50 µM)) such that 35 μl contain 500 TSP.
3. Place a 35 μl drop of cell suspension every other well of a 60 well Terasaki plate.
   NOTE: 35 μl drops can fuse if they are placed in contiguous wells.
4. Place one irradiated lobe on the surface of each drop. Close the Terasaki plate and turn the plate upside down to let the cells reach the apical pole of the drop by gravity.
5. Hit gently the upper side of the inverted plate to force the lobes to slide to the apical edge of the drop. Incubate the inverted Terasaki plate in a humidified incubator for 48 hr at 37 °C + 5% CO₂. After colonization, either culture E14 thymi in FTOC⁷, or graft under the kidney capsule of a recipient adult mouse ⁸.

7. Fetal Thymic Organ Culture

1. Place 3 ml of complete medium on a 35 mm Petri dish. Place an isopore membrane filter floating on the medium. Place the reconstituted lobes on the edge of the membrane with a forceps (no more than 8 lobes on one membrane).
2. Place the Petri dishes containing the thymic lobes inside a 90 mm Petri dish, together with a 35 mm dish, without cover, containing 2 ml of water, to ensure optimal humidity. Incubate 12 days at 37 °C + 5% CO₂.

8. Grafts Under the Kidney Capsule Under Sterile Conditions

NOTE: The kidney parenchyma is surrounded by connective tissue forming a capsule. The sub-capsular region is particularly rich in blood and lymphatic vessels thereby providing a suitable environment for the development of grafts (e.g. thymic lobes, pancreatic islets or newborn hearts). Grafts are usually done on the left kidney because it is more accessible than the right kidney. CD3^-/- male mice were used as recipients thus avoiding graft versus host reaction due to minor histocompatibility antigens linked to the Y chromosome because sex determination before E15 is not easily done.

1. Sterilize the instruments and wear sterile gloves along the surgery. Anesthetize the mouse by intra peritoneal injection of a solution of Ketamine 10 mg/ml + Xylazine 1 mg/ml diluted in PBS: (50 to 100 μl per 10 g body weight), using a 1 ml syringe. Verify anesthesia by checking lack of interdigital reflexes. Place a drop of hydro-Optimune gel on each eye to prevent dryness during anesthesia.
   NOTE: The solution should be prepared extemporaneously and warmed at room temperature before injection. The anesthesia lasts at least 20 min. The degree of anesthesia should be controlled all along the surgery. The anesthesia can be prolonged by intra peritoneal injection of half dose every 20 min.
2. Place the mouse on its right side, under a horizontal lamina flow hood. Sterilize the surgical field with 70% ethanol and 10% iodide dermic solution. With a forceps part the mouse hair along a 2 cm length just above the joint of the hind leg. Shave the surgical area.
3. With a scalpel, make an incision 1.5 cm length of first, the cutaneous tissue, then with scissors cut the muscular tissue. Apply a slight pressure to both sides of the incision, which will force the kidney out of the abdominal cavity.
4. Apply saline with a cotton-bud to keep the kidney moist. If you have difficulties in exposing the organ, use a flat forceps to pull out the surrounding adipocyte tissue to which the kidney is attached. Maintain the kidney out of the retroperitoneal cavity by a cotton bud placed underneath the organ.
5. With two fine forceps, make a 2-3 mm hole in the capsule (avoid touching the parenchyma to prevent bleeding). During the whole surgical procedure keep the exposed kidney capsule continuously moistened.
6. Insert carefully the lobes under the kidney capsule, by maintaining the wall of the capsule opened and slide the graft under the capsule towards the edge pole. Place up to 6 lobes per kidney.
7. Replace the kidney in the retro-peritoneal cavity. Suture the muscle aponeuroses, then the skin with individual surgeon knots. Wet the wound with 10% iodide povidone solution. Place the mouse on a heated pad at 37 °C.
8. Survey the transplanted mouse until full recovery from anesthesia by control of cardiac, respiratory and whisker movements, mucosal colors until total recovery seen by self contained movements.
   NOTE: We recommend injection of analgesic solution (Buprenorphin, 0.1 mg/kg) intra muscle just after the surgery and the 2 days following surgery to prevent post-surgery pain.
9. Keep the operated animal in isolation until fully recovered. Inspect every other day the stitches of the wound to verify and prevent infection. Never observed wound infection following this procedure.
   NOTE: Weekly analysis of peripheral blood cells from the CD3^-/- grafted mice allow us to detect the thymic derived populations originated from either E13 or E18 progenitors using the CD45 allotypic marker and T cell lineage specific antibodies (Figure 3).

9. Analysis of the TSP progeny by Flow Cytometry

1. Tease the FTOCs individually to obtain single cell suspensions (see protocol section 4).
2. Stain cell suspensions from individual lobes with an antibody mixture containing antibodies recognizing CD45.1 PECy7, CD45.2 AmCyan, CD4 APCCy7, CD8 APC, CD3 Pacific Blue, Vγ5 FITC, Vδ1 PE and incubate as described in section 5.
3. Re-suspend the cells in HBSS + 1% FCS + propidium iodide and analyze in a flow cytometer (see results in Figure 2).

Wyniki

In order to choose a method to deplete thymic lobes of endogenous thymocytes allowing the best development of colonizing progenitors, we compared the levels of T cell reconstitution in thymic lobes colonized after irradiation or a 5-day deoxy-guanosine (d-Gua) treatment. The results show that while there is no difference at day 9 of culture, irradiated lobes contained more T cells than those treated with d-Gua, at day 12. Thus, irradiation is more appropriate than d-Gua treatment to obtain T cell development after thymic...

Dyskusje

Two main assays can be used to analyze T cell differentiation ex vivo. The most recently reported is the co-culture of hematopoietic progenitors with BM stromal cells, OP9, expressing the ligands of Noth1, delta like 1 or 4¹². This 2-D assay is easy to perform, highly efficient and sensitive, allowing analysis at the single cell level. However, it neither supports T cell development beyond the stage of DP nor the generation of γδ DETC¹³, both of which require direct interactions w...

Materiały

Name	Company	Catalog Number	Comments
90 x 15 mm and 35 x 15 mm plastic tissue culture petri dishes.	TPP	T93100/T9340	Sampling
26GA 3/8 IN 0.45x10mm syringes with needles Beckton-Dickinson Plastipak.	BD Plastipak	300015	Cell suspension
Nylon mesh bolting cloth sterilized 50/50 mm pieces.	SEFAR NITEX	03-100/32	Filtration of cells
Ethanol 70%.	VWR	83801.36	Sterility Actions
Iodide Povidone 10% (Betadine)	MEDA Pharma	314997.8	Sterility Actions
Ketamine 100mg/ml (stock solution)	MERIAL	-	Anesthesic
Xylazine 100mg/ml in PBS (stock solution)	Sigma	X1251-1G	Anesthesic and muscle relaxant
Buprenorphin 0.3mg/ml stock solution	AXIENCE	-	Morphinic analgesic
Ophtalmic gel (0.2% cyclosporin)	Schering-Plough Animal Health	-	Eyes protection
DPBS (+ CaCl2, MgCl2)	GIBCO Life Technology	14040-174	to isolate embryos
HBSS Hanks' Balanced Salt Solution (+ CaCl2, MgCl2)	GIBCO Life Technology	24020-091	to wash out the blood and dissect the embryos
OPTI-MEM I GlutaMAX I	GIBCO Life Technology	51985-026	Medium for cultures
Fœtal Calf serum	EUROBIO	CVFSVF00-0U	additive for cultures
Penicilin and Streptomycin	GIBCO Life Technology	15640-055	Antibiotics for cultures
2 b mercapto ethanol	GIBCO Life Technology	31350010	additive for cultures
LEICA MZ6 Dissection microscope	LEICA	MZ6 10445111	Occular W-Pl10x/23
Cold lamp source	SCHOTT VWR	KL1500 compact	Two goose neck fibers adapted
Silicone elastomer	World Precision Instruments	SYLG184	Dissecting Pad
Spoon, round and perforated	Fine Science Tools	10370-18	Dissection tools
Fine Iris Scissors	Fine Science Tools	14090-09	Dissection tools
Vannas spring Scissors	Fine Science Tools	15018-10	Dissection tools
Forceps : Dumont #5/45 Inox 11 cm	F.S.T.	11253-25	Dissection tools
Two pairs of fine straight watchmakers’ forceps Dumont #5 11 cm fine tips.	Fine Science Tools	11295-20	Dissection tools
Polyamid thread with needle 6-0   C-3 3/8c	ETHICON	F2403	for sutures
Needle-holder	MORIA/F.S.T.	12060-02	for sutures
Heating pad	VWR	100229-100	To maintain mouse temperature during anesthesy
Membrane Isopore RTTPO2500	DUTSCHER	44210	For FTOC
Terasaki 60 wells plates	FISHER	1x270 10318801	For hanging drop technique
Gauze swabs steriles 7.5cmx7.5cm	Hydrex	11522	To apply disinfecting solution
Fluorescence or biotin labelled antibodies	BD Biosciences, Biolegend or e-Biocsiences	Clone Number see table below	Staining cells
MACS Columns/Streptavidin Microbeads	Miltenyi Biotec	130-042-401/130-048-101	Cell depletion
Mice	Charles Rivers Laboratories (CD45.1) and Janvier Labs  (CD45.2)	C57BL/6 CD45.1 OR CD45.2	Source of cells and thymic lobes
Mice	CDTA Orléans, France	CD 3 epsilon Ko CD45.2	Grafting experiment