Method for generating T cells progenitors

Abstract

The invention relates to an in vitro method to generate T cell progenitors, comprising the step of culturing CD34+ cells in a medium containing TNF-alpha and/or an antagonist of the Aryl hydro-carbon/Dioxin receptor, in particular StemRegenin 1 (SR1), in presence of a Notch ligand and optionally a fibronectin fragment.

Claims

1. An in vitro method for generating T cell progenitors having the phenotype CD34-CD7+, comprising culturing CD34+ cells in a culture medium comprising fetal serum, TNF-alpha, IL-7, thrombopoietin (TPO), Flt3L, and Stem cell factor (SCF) and not comprising IL-3, in the presence of an immobilized Delta-like-4 ligand and a fibronectin fragment for a period of more than 5 days and less than 10 days, wherein said fibronectin fragment comprises the RGDS (SEQ ID NO: 3) and connecting segment 1 (CS-1) motifs as well as a heparin-binding domain, wherein TNF-alpha is present in the culture medium at a concentration of at least 10 ng/ml, wherein fetal serum is present in the culture medium at a concentration of at least 15%, and wherein the CD34-CD7+ cells represent more than 60% of the CD7+cell population.

2. The method of claim 1, wherein the Delta-like-4 ligand is immobilized on an inner surface of a culture vessel or on a surface of beads present in the culture medium.

3. The method of claim 1, wherein the CD34+ cells have been isolated from an adult donor.

4. The method of claim 1, wherein the Delta-like-4 ligand is the soluble domain of the Delta-like-4 ligand fused to an Fc region of an IgG protein.

5. The method of claim 1, wherein the fibronectin fragment is immobilized on an inner surface of a culture vessel or on beads.

6. The method of claim 1, wherein the fibronectin fragment is CH-296.

7. The method of claim 1, wherein the CD34-CD7+ cells are also CD5-.

8. The method of claim 1, wherein the CD34-CD7+ cells are also CD1a-.

9. The method of claim 7, wherein the CD34-CD7+CD5- cells are also CD1a-.

10. The method of claim 1, wherein the CD34+ cells are isolated from a human.

11. The method of claim 1, further comprising purifying the generated T cell progenitors.

12. The method of claim 11, further comprising conditioning the T cell progenitors in a pouch for injection into a patient.

13. The method of claim 1, wherein greater than 80% of the CD7+ cells in the T cell progenitor population are CD34-CD5-, CD34-CD1a-, and/or CD34-CD1a-CD5-.

14. The method of claim 1, wherein the CD34-CD7+ cells represent more than 70% of the CD7+ cell population.

15. The method of claim 1, wherein the CD34-CD7+ cells represent more than 90% of the CD7+ cell population.

16. The method of claim 8, wherein the CD34-CD7+CD1a- cells represent more than 70% of the CD7+ cell population.

17. The method of claim 1, wherein the medium comprises at least 20% of fetal serum.

18. The method of claim 1, wherein the fetal serum is fetal bovine serum or fetal calf serum.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Total nucleated cells number obtained starting with 20000 CD34+ cells from cord blood (CB, FIG. 1.A) or mobilized peripheral blood (mPB, FIG. 1.B), after 3 days (black bars) and 7 days (cumulative black and grey bars) of culture. NC: not complemented; SR1: addition of StemRegenin 1 (750 nM); TNF-alpha: addition of TNF-alpha (100 ng/ml); +(a), (b), (c): number of cells observed when the complements are added from 0-3 days of culture (a), 0-7 days of culture (b) or 4-7 days of culture (c).

(2) FIG. 2: Number of CD7+ T-cell precursors obtained at day 7 starting with 20000 CD34+ cells from cord blood (CB, FIG. 2.A) or mobilized peripheral blood (mPB, FIG. 2.B). Black bars: CD34+CD7+ cells; grey bars: CD34-CD7+ cells. (a), (b), (c): number of cells observed when the complements are added from 0-3 days of culture (a), 0-7 days of culture (b) or 4-7 days of culture (c).

(3) FIG. 3: Expression of BcI11b was analyzed on lived cells after 7 days of culture obtained starting with CD34+ cells from cord blood (CB) or mobilized peripheral blood (mPB) cultured in the presence (grey bars) or absence (black bars) of TNF-alpha.

(4) FIG. 4: Combined effect of TNF-alpha and the Notch ligand DL4 on the number of CD7+ cells obtained at day 7 starting from CD34+ cord blood cells (CB, black bars, left) or mobilized peripheral blood (mPB, grey bars, right). (+/−means presence or absence of DL4 or TNF-alpha).

(5) FIG. 5: Frequency of myeloid cells at day 7 obtained starting from CD34+ cells from cord blood (CB) or mobilized peripheral blood (mPB) in presence (grey bars) or not (black bars) of TNF-alpha (Mean±SEM).

(6) FIG. 6: Total CD7+ cells number obtained from day 3 to day 7 in a dose response assay of TNF-alpha, starting with CD34+ cells from cord blood (CB, FIG. 6.A) or mobilized peripheral blood (mPB, FIG. 6.B).

(7) FIG. 7: Proportion of CD34-CD7+ cells (grey bars) vs CD34+CD7+ cells (black bars) in a dose response assay of TNF-alpha starting with CD34+ cells from cord blood (CB, FIG. 7.A) or mobilized peripheral blood (mPB, FIG. 7.B)

(8) FIG. 8: Proportion of cells in the different phases of the cellular cycle. A. CB: cells differentiated from cord blood; B. cells differentiated from mobilized peripheral blood cells. NC: non complemented; TNF-alpha: cultured in presence of TNF-alpha (20 ng/ml); SR1: cultures in presence of SR1 (30 ng/ml)

(9) FIG. 9: percentage (A) and total number (B) of CD5+CD7+ cells cultured starting with CD34+ cells from cord blood in presence of TNF-alpha and/or SR1 at various concentrations, after 7 days of culture.

EXAMPLES

Example 1—Material and Methods

(10) Human Cells

(11) Cord blood samples not eligible for banking were used for research purposes, following the provision of informed consent by the child's mother. Mobilized Peripheral Blood (mPB) samples were collected from healthy donors after G-CSF mobilization. Samples were directly enriched for CD34+ cells. The informed consent was given by each donor (Biotherapy Department, Necker Hospital, Paris).

(12) Exposure of CD34+ Progenitor Cells to Notch Ligand DL-4

(13) CD34+ cells from human CB or mobilized peripheral blood samples were cultured in 24-well plates or 6-well plates that had been coated with recombinant human fibronection (RetroNectin®, Clontech/Takara) and DL-4 (5 μg/ml, PX'Therapeutics, Grenoble, France). Coating was performed for 2 h at 37° C., DL-4 coated wells were then blocked with bovine serum albumin 2% (BSA) in phosphate-buffered saline (PBS) for 30 minutes at 37° C. and washed with PBS. Cultures were initiated at a concentration of 2×10.sup.4 cells/well or 1×10.sup.5 cells/well (for 24-well and 6-well plates respectively) in α-MEM medium (Gibco, life Technology), supplemented with NaHCO.sub.3(7.5%) (Gibco, life Technology) and 20% defined fetal calf serum (Hyclone, Thermo Fisher Scientific, Illkirch, France) and the recombinant human cytokines interleukin-7 (IL-7), Flt3-ligand (Flt-3), stem cell factor (SCF) and thrombopoietin (TPO) (all at 100 ng/ml and all purchased from PeproTech Inc, Rocky Hill, N.J.) with or without TNF-α (R&D Systems, US). After 3 days of culture, the cells were half replaced by fresh medium. Cultured cells were analyzed by fluorescence-activated cell sorting (FACS) after 3 and 7 days of culture on DL-4 respectively to exclude CD34-/CD7− myeloid cells from subsequent analyses.

(14) In Vitro T Cell Differentiation Assay on OP9/DL1 Cells

(15) The T-lymphoid potential of native CD34+CB cells and TNF-α induced T cell progenitors generated by exposure to DL-4 was assessed in OP9/DL-1 co-cultures, as previously described (Six et al, Blood Cells Mol Dis. 2011 Jun. 15; 47(1):72-8 and Six J Exp Med. 2007 Dec. 24; 204(13):3085-93).

(16) Quantitative, Real-Time Polymerase Chain Reactions Using RT2 Profiler Array

(17) CD7+ cells were sorted on Ariall after 7 days of culture. Total RNA of sorted cell fractions from day 3 and day 7 was isolated with the Rneasy Micro Kit (Qiagen, Courtaboeuf, France). RT2 Profiler PCR arrays were performed following the protocol detailed in the RT2 Profiler PCR Array Handbook (SA Biosciences, Frederick Md.).

(18) Flow Cytometry Analysis and Cell Sorting

(19) Monoclonal antibodies against human CD34 (AC136), CD3 (BW264/56), CD45 (5B1) were purchased from Miltenyi Biotech (Bergisch Gladbach, Germany), and CD4 (SK4), CD7 (M-T701), CD25 (M-A251), 7-aminoactinomycin D (7AAD) were from BD Biosciences (San José, Calif.). Anti-human CD8 (RPAT8) was from Sony Biotechnology (San Jose, USA). The Anti-human Ctip2 (BcI11b) antibody was from Abcam (Cambridge, UK).

(20) Human cells were stained and analyzed using a Gallios analyzer (Beckman Coulter, Krefeld, Germany). Cells from xenogenic recipients were analysed on a MACSQuant® apparatus (Miltenyi Biotech, Bergisch Gladbach, Germany). The data were analyzed using FlowJo software (Treestar, Ashland, Oreg.) after gating on viable, 7AAD-negative cells. Cell subsets were sorted on an ARIA II system.

(21) Cell Proliferation Assays

(22) For cell proliferation assays, CD34+ cells from CB and mPB were labeled using the CellTrace™ CFSE kit (Life Technologies, Carlsbad, Calif.) prior to culture with DL-4 and TNF-alpha (Life Technologies). The cells' staining intensity was measured prior to culture each day from day 3 to day 7. CFSE-positive cell were analyzed on a Gallios cytometer (Beckman Coulter).

(23) Cell Cycle Assays

(24) For cell cycle analysis, cells were stained with Hoechst33342 (Life technology) and Ki67-PC5 (BD Bioscience) after fixed with Fixative reagent of PerFix-nc kit (Beckman Coulter) at room temperature for 15 min, and added permeablizing reagent. The data were analyzed using FlowJo software (version 10.2, Treestar, Ashland, Oreg.) after gating on viable, 7AAD-negative cells.

(25) Adoptive Transfer of In Vitro-Generated T-Cell Progenitors Derived from Adult HSPCs into NSG Neonates

(26) All experiments and procedures with animals were performed in compliance with the French Ministry of Agriculture's regulations on animal experiments. The injection of in vitro generated human T-cell progenitors in NSG mice has been approved by the Ministry of Higher Education and Research (APAFIS 2101-2015090411495178v4).

(27) The NSG (NOD-Scid(IL2Rg.sup.null)) mice (obtained from the Jackson Laboratory, Bar Harbor, Me., http://www.jax.org) were kept in a pathogen-free facility. Progeny derived from mPB CD34+ HSPCs in 7-day DL-4 cultures with or without TNF a (3×10.sup.5 or 1×10.sup.6) were injected intra-hepatically into NSG neonates (0-4 days old). Control mice were injected with either 3×10.sup.5 non-cultured mPB CD34+ cells or 100 ul PBS.

(28) Average engraftment levels of NSG mice were determined from 4 to 12 weeks post-transplant. Flow cytometry analysis was performed on freshly cells collected from femur, thymus, peripheral blood and spleen. Cells were treated with 1× red blood cell lysis buffer (Biolegend, US) and washed before stained by antibodies.

(29) Analysis of T Cell Receptor Diversity

(30) TCR gene rearrangement analysis was performed in duplicate and on the two independently purified subsets (average is shown).

(31) TCR-δ quantification (D δ2-D δ3, D δ2-J δ1, and D δ3-J δ1) was performed with the listed sets of primers and probes.

(32) The following were used for D δ2-D δ3 rearrangements:

(33) TABLE-US-00001 D δ2, (SEQ ID No 9) 5′-CAAGGAAAGGGAAAAAGGAAGAA-3′; D δ3, (SEQ ID No 10) 5′-TTGCCCCTGCAGTTTTTGTAC-3′; and D′3 probe, (SEQ ID No 11) 5′-ATACGCACAGTGCTACAAAACCTACAGAGACCT-3′.

(34) The following primers and probe were used for D δ2-J δ1 rearrangements:

(35) TABLE-US-00002 D δ2, (SEQ ID No 12) 5′-AGCGGGTGGTGATGGCAAAGT-3′; J δ1, (SEQ ID No 13) 5′-TTAGATGGAGGATGCCTTAACCTTA-3′; and J δ1 probe, (SEQ ID No 14) 5′-CCCGTGTGACTGTGGAACCAAGTAAGTAACTC-3′

(36) The following were used for D δ3-J δ1 rearrangements:

(37) TABLE-US-00003 D δ3, (SEQ ID No 15) 5′-GACTTGGAGAAAACATCTGGTTCTG-3′; J δ1 primer and J δ1 probe.

(38) The analysis of TCR rearrangements by multiplex fluorescent PCR was performed by separation of fluorochrome-labeled single stand PCR products in a capillary sequencing polymer and detected via automated laser scanning.

(39) Apoptosis Assays

(40) Cells were washed by cold PBS and resuspended in binding buffer at a concentration of 1 million cells/ml. After adding 5 ul Annexin V-PE (BD Bioscience) and 2 ul 7AAD, cells were incubated in the dark at room temperature for 15 min. Subsequently, cells were washed with 500 ul binding buffer and resuspended in 100 ul binding buffer to be analyzed within 1 hour.

Example 2: Improvement on the Expansion and Differentiation of T-Cell Precursors

(41) When CD34+ cells are cultured with DL-4, FIG. 1 shows that addition of TNF-alpha to the cell culture medium makes it possible to multiply the total number of cells recovered at day 7 by 10 times as compared to culture without TNF-alpha, either when starting with CD34+ cells issued from cord blood (FIG. 1.A) or from PB (FIG. 1.B).

(42) FIG. 2 shows that addition of TNF-alpha to the cell culture medium makes it possible to multiply the number of CD7+ cells in by 20 to 40 times, either when starting with CD34+ cells issued from cord blood (FIG. 2.A) or from PB (FIG. 2.B). The improvement is especially high for the CD34-CD7+ cell population.

Example 3: Analysis of the Surface Markers of the T Cell Progenitors

(43) The surface markers present at the surface of the cells obtained after 7 days of culture were determined by flow cytometry.

(44) TABLE-US-00004 Cord blood mPB CD34+ CD34− CD34+ CD34− CD34+ CD34− CD34+ CD34− CD7+ CD7+ CD7− CD7− CD7+ CD7+ CD7− CD7− −TNFα 29.5 38.9 15.4 16.3 15.1 11.3 32.9 40.7 +TNFα 0.39 95.6 0.76 3.22 0.68 90.1 2.75 6.49

(45) TABLE-US-00005 Cord blood mPB CD5+ CD5− CD5+ CD5− CD5+ CD5− CD5+ CD5− CD7+ CD7+ CD7− CD7− CD7+ CD7+ CD7− CD7− −TNFα 1.56 66.8 5.10.sup.−3 31.6 0.069 26.4 0.027 73.5 +TNFα 0.66 95.4 0.012 3.97 3.01 87.7 0.24 9.00

(46) These tables show that addition of TNF-alpha leads to an increase in the proportion of CD7+ cells, without really increasing the proportion of CD5+ cells.

(47) After 7 day culture, HSPCs differentiate into CD34-CD7+CD5− T-cell precursors.

(48) The surface markers present at the surface of the cells obtained after 10 days of culture were also determined by flow cytometry.

(49) No expression of CD1a was found (data not shown).

(50) The kinetics of modification of the surface markers was studied and it was found that presence of TNF-alpha in the culture medium increases the proportion of CD7+ from day 4 up to day 7 (data not shown).

Example 4: Rearrangement of T Cell Receptors

(51) DL-4 T-cell precursors do not exhibit any signs of TCR rearrangement with or without TNF-alpha or SR1 after 7 days of culture (data not shown).

(52) Specific analysis of rearrangement was performed:

(53) Results of TCRdelta Rearrangements

(54) Detection of Dδ2-Dδ3 rearrangements in CB-NC et CB-SR1. No other TCRdelta rearrangements were detected. Results were in accordance with RQ-PCR quantification

(55) Results of TCRgamma Rearrangements

(56) No TCRgamma rearrangements were detected.

(57) Results of TCRbeta rearrangements

(58) No TCRbeta rearrangements were detected.

Example 5: T-Commitment of TNF a Induced T-Cell Precusors

(59) BcI11b is an important transcriptional factor uniquely switched on since T-cell commitment and absolutely required for T-cell differentiation.

(60) Intracellular staining on T-cell precursors cultured with TNF-alpha showed positive expression of BcI11b for both CD34+ cells issued from cord blood, and mPB. FIG. 3 shows that TNF-alpha increases the proportion of total cells expressing BcI11b transcription factor. When cultured with TNF-alpha, the proportion of BcI11b expressing cells was increased.

Example 6: Differentiation on Other Lineages

(61) Presence of other cell surface markers (CD14 and CD33) specific of other lineages was assessed.

(62) FIG. 5 shows that the culture in the presence of TNF-alpha made such cells not detectable, whereas their proportion is less than 22% when CD34+ cells are cultured without TNF-alpha.

Example 7: TNF-Alpha Reduces Apoptosis of the Cells

(63) Apoptosis markers (7AAD and Annexin5) were studied.

(64) TABLE-US-00006 7AAD +/− AnnexinV + apoptotic cells (%) CB mPB −TNFα 1.66 10.56 +TNFα 0.28 0.96

(65) This table shows that culture in presence of TNF-alpha reduces the presence of the apoptosis markers. This is particularly apparent for mPB.

Example 8: Dose Response Assay of TNF-Alpha

(66) Various doses of TNF-alpha were used.

(67) FIG. 6 shows that TNFa may increase CD7+ cell numbers during culture, when the concentration is more than 10 ng/ml, either for CB cells (FIG. 6.A) or mPB cells (FIG. 6.B).

(68) Kinetics of the dose response showed that TNF-alpha increases the T-cell differentiation after only 4 days of culture in DL-4. There was no difference between the concentration 10, 50 and 100 ng/ml (not shown).

(69) To determine the threshold of effective concentration, analysis of lower concentrations (0.01-10 ng/ml) was performed.

(70) The effect of TNF-alpha on CB and mPB on T-cell differentiation (percentage of CD34− CD7+ cells) was found to be concentration-dependent at low concentration (FIG. 7). The total number of CD7+ T-cell precursors was not different from 5 ng/ml to 100 ng/ml.

Example 9: Proliferation Analysis During Culture

(71) TNF-alpha was found to increase the proliferation of CD34+CD7+ T-cell precursors since day 3 in DL-4 culture as compared to culture conditions without TNF-alpha (data not shown).

Example 10: Synergy Between TNF-Alpha and the Notch Ligand

(72) FIG. 4 shows that without DL4, both CB and mPB failed to differentiate into CD7+ T-cell precursors. Even the complementation of the medium with TNF-alpha couldn't rescue it.

(73) When both TNF-alpha and the Notch ligand are present, the effect observed is very high. It thus seems that there is a synergy between these two compounds and that the effect of TNF-alpha on T-cell differentiation is likely Notch dependant.

Example 11: Addition of TNF-Alpha Increases Proliferation of CD7+ Progenitors

(74) After 7 days of culture in presence of TNF-alpha, CD34+CD7-, CD34+CD7+ and CD34-CD7+ subsets were sorted, stained with CFSE (Carboxyfluorescein succinimidyl ester) and the dilution of CFSE (surrogate marker of cell proliferation) was followed from day 8 to 10.

(75) Only CD34+CD7+ and CD34-CD7+ cells show increased proliferation when cultured with TNF-alpha. (Data not shown)

Example 12: Cell Cycle Analysis

(76) Analysis of the cell cycle was performed. It was observed that more cells were released from GO phase in presence of TNF-alpha on both CB and mPB derived CD7+ progenitors (FIG. 8).

Example 13: Combination of SR1 and TNFa

(77) SR1 accelerates T-cell differentiation as shown by the presence of CD5+CD7+ cells at day 7. The number of CD5+CD7+ cells is increased by the presence of both TNF-alpha and SR1 (FIG. 9).

Example 14: In Vivo Data

(78) T-cell precursors induced in presence of TNF-alpha may largely fasten the reconstitution of the T-lineage in vivo.

(79) Indeed, 4 weeks post-transplantation, recipient mice injected with mPB T-cell precursors produced in the presence of TNF-alpha have larger thymus than mice injected with mPB T-cell precursors produced without TNF-alpha. T-cell precursors induced in presence of TNF-alpha can differentiate to activated TCRαβ T cells within 4 weeks in vivo. (Data not shown)

(80) In summary, addition of TNF-alpha from day 0 in the DL-4 culture system leads to an increase of T-cell progenitors (defined by the surface expression of CD7) of 40 fold for mPB HSPC and 20 fold for CB HSPC at day 7.

(81) The CD7+ T-cell progenitors generated from both CB and mPB were mostly CD34− and were CD1a negative. Cells were also mostly CD5 negative.

(82) They expressed BcI11b, which is important fine-turning molecular for T-commitment and further T-cell differentiation.

(83) They did not exhibit any signs of T-cell receptor rearrangements.

(84) Their phenotype and molecular characteristics were similar to the one of the CD34-CD7+ T-cell progenitors obtained without TNF-alpha.

(85) Regarding the mechanisms involved in TNF-alpha action, TNF-alpha decreases expression of apoptosis markers and increases cell proliferation during the culture. It also inhibits myeloid cell production.

(86) The use of TNF-alpha in the DL4 culture system increase to a huge extent the amounts of T-cell progenitors produced from both human adult and cord blood HSPC. It may thus overcome the difficulty to obtain large amounts of T-cell progenitors from adult HSPC. It may also decrease the number of starting HSPC required in future clinical trials and the quantity of GMP grade and other reagents required, thus decreasing the costs of production of these T-cell progenitors.