Use of common gamma chain cytokines for the visualization, isolation and genetic modification of memory T lymphocytes
11395835 · 2022-07-26
Assignee
Inventors
Cpc classification
A61P35/00
HUMAN NECESSITIES
A61P31/00
HUMAN NECESSITIES
G01N33/6863
PHYSICS
A61K35/17
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61P37/06
HUMAN NECESSITIES
A61K48/0091
HUMAN NECESSITIES
C12N2501/51
CHEMISTRY; METALLURGY
International classification
A61K35/17
HUMAN NECESSITIES
A61K48/00
HUMAN NECESSITIES
Abstract
It is described in vitro methods for expanding, detecting or isolating rare populations of antigen specific memory T cells. It is also described an in vitro method for obtaining a genetically modified memory T cell population. Uses of cells so obtained are also disclosed.
Claims
1. A method of treating or preventing graft versus host disease (GvHD), in a patient in need thereof, wherein the GvHD results from administering a genetically modified T cell population, that is CD4+ and/or CD8+, to a patient in need thereof, the method comprising administering an amount of ganciclovir effective to treat GvHD to the patient in need thereof; the genetically modified T cell population, that is CD4+ or CD8+, having been prepared by the steps comprising: a) activating lymphocytes in vitro in a cell-free medium with at least two specific activating receptor agonist antibodies that are able to drive lymphocyte activation, wherein one of the lymphocyte activating receptor agonist antibodies is specific for CD3 polypeptide and the other lymphocyte activating receptor agonist antibody is specific for CD28; b) exposing activated lymphocytes in vitro, in a cell-free medium, to an effective amount of interleukin added to the medium, wherein the interleukin is at least IL-7 and IL-15, able to selectively expand populations of memory T cells; and c) inserting and expressing at least one exogenous gene by means of an appropriate vector, into the lymphocytes as obtained in b) to produce a genetically modified memory T cell population that is CD4+ or CD8+; wherein the at least one exogenous gene comprises a thymidine kinase suicide gene; wherein the effective amount of the interleukin added to the medium is from 5 ng/ml to 50 ng/ml; wherein the medium and interleukin of step b) are replaced every 3 to 4 days; and wherein about 80% of both CD8+ and CD4+ T-cells of steps b) and c) are positive for a CD62L marker and a CD127 marker, and the genetically modified T cell population further comprises central memory T-lymphocytes; so that GvHD is treated or prevented.
2. The method of claim 1, wherein the lymphocytes of step (a) are derived from a biological sample selected from the group consisting of: blood and other liquid samples of biological origin, solid tissue samples, tissue cultures of cells derived therefrom and the progeny thereof, and isolated cells from biological samples.
3. The method of claim 1, wherein the specific lymphocyte activating receptor agonist of step (a) is conjugated to cell-mimicking cell-free supports.
4. The method of claim 3, wherein the cell-mimicking supports are paramagnetic beads.
5. The method of claim 1, wherein the vector of step (c) is a viral vector.
6. The method of claim 1, wherein the at least one exogenous gene of step (c) further comprises a gene selected from the group consisting of a marker gene, a biologically active molecule, a receptor, a soluble factor retained in the cell or released outside the cell, a gene conferring resistance to a prodrug and combinations thereof.
7. The method of claim 1, wherein the effective amount of the interleukin added to the medium is 5 ng/ml or 50 ng/ml.
8. The method of claim 1, wherein the genetically modified T cell population, that is CD4+ and/or CD8+, is administered to the patient for treating a cancer in the patient.
9. The method of claim 8, wherein the cancer is an adenocarcinoma.
10. The method of claim 8, wherein the cancer is a mammary adenocarcinoma.
11. A method of treating cancer, comprising administering a genetically modified T cell population, that is CD4+ or CD8+, to a patient in need thereof, the genetically modified T cell population, that is CD4+ or CD8+, having been prepared by the steps comprising: a) activating lymphocytes in vitro in a cell-free medium with at least two specific activating receptor agonist antibodies that are able to drive lymphocyte activation, wherein one of the lymphocyte activating receptor agonist antibodies is specific for CD3 polypeptide and the other lymphocyte activating receptor agonist antibody is specific for CD28; b) exposing activated lymphocytes in vitro, in a cell-free medium, to an effective amount of interleukin added to the medium, wherein the interleukin is at least IL-7 and IL-15, able to selectively expand populations of memory T cells; and c) inserting and expressing at least one exogenous gene by means of an appropriate vector into the lymphocytes as obtained in b) to produce a genetically modified memory T cell population that is CD4+ or CD8+; wherein the at least one exogenous gene comprises a thymidine kinase suicide gene; wherein the effective amount of the interleukin added to the medium is from 5 ng/ml to 50 ng/ml; wherein the medium and interleukin of step b) are replaced every 3 to 4 days; and wherein about 80% of both CD8+ and CD4+T-cells of steps b) and c) are positive for the marker CD62L and marker CD127 and are central memory T-lymphocytes; and administering an effective amount of ganciclovir to the patient, when the patient manifests graft versus host disease (GvHD) as a result of the administration of the genetically modified T cell population, that is CD4+ or CD8+.
12. The method of claim 11, wherein the lymphocytes of step (a) are derived from a biological sample selected from the group consisting of: blood and other liquid samples of biological origin, solid tissue samples, tissue cultures of cells derived therefrom and the progeny thereof, and isolated cells from biological samples.
13. The method of claim 11, wherein the specific lymphocyte activating receptor agonist of step (a) is conjugated to cell-mimicking cell-free supports.
14. The method of claim 13, wherein the cell-mimicking supports are paramagnetic beads.
15. The method of claim 11, wherein the vector of step (c) is a viral vector.
16. The method of claim 11, wherein the exogenous gene of step (c) comprises a gene selected from the group consisting of a suicide gene, a marker gene, a biologically active molecule, a receptor, a soluble factor retained in the cell or released outside the cell, a gene conferring resistance to a prodrug and combinations thereof.
17. The method of claim 11, wherein the effective amount of the interleukin added to the medium is 5 ng/ml or 50 ng/ml.
18. The method of claim 11, wherein the cancer is an adenocarcinoma.
19. The method of claim 11, wherein the cancer is a mammary adenocarcinoma.
Description
(1) The invention will be now described by means of non-limiting examples, making reference to the following figures:
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(37) Transduced T cells generated either with beads conjugated with anti-CD3 and anti-CD28 and cultured with IL-7 or IL-2, or by activation with soluble anti-CD3 and culture in IL-2 were stained with CFSE at day 9 after initial stimulation, and were co-cultured with irradiated allogeneic PBMCs. Unmanipulated peripheral blood lymphocytes (PBL) from the same donors were stained with CFSE, co-cultured with the same irradiated allogeneic PBMCs and used as controls. After 7 days cells were counted, stained with To-pro3, and analysed by FACS to evaluate the percentage of dividing and/or dying cells. A) Percentage of dividing cells. B) Total number of dying cells. Activation induced cell death was calculated on dividing cells (white, lower part of histograms) and death for neglect was calculated on non-dividing cells (black, upper part of histograms). *=p<0.05 **=p<0.01.
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(39) A) Cells treated as described in
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(41) NOD/scid mice were conditioned and transplanted with human skin, were infused i.v. with 20×10.sup.6 transduced lymphocytes generated with beads CD3/CD28 and culture with IL7 or IL2, or with OKT3 and culture with IL-2. Human chimerism was assessed weekly by flow-cytometry after staining for human CD3 and mouse CD45. Quadrants and percentages were set according to isotype control staining. Kinetic of human chimerism is shown (n=4 donors).
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(43) NOD/scid mice were conditioned and transplanted with human skin, were infused i.v. with 20×10.sup.6 transduced lymphocytes generated with beads CD3/CD28 and culture with IL7 or IL2, or with OKT3 and culture with IL-2. Mice were monitored for xenogenic GvHD according 1) Weight loss. 2) GvHD clinical score (described in material and methods). Controls: animals that did not receive infusions of lymphocytes.
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(45) Three weeks after human T-cell infusion, NOD/Scid chimera mice were sacrificed and human skin was removed bilaterally. All biopsies were subsequently blindly analysed by pathologists through ematossilin-eosin (EE) and anti human CD3 staining (αhCD3). Representative sections are reported.
MATERIALS AND METHODS
(46) Experimental protocols were approved by the Ethical Committee of the San Raffaele Scientific Institute and performed according to its guidelines.
(47) Mice and Tumor Cells
(48) Seven to 8-week old BALB/c and CD45.2.sup.+ C57BL/6 mice were purchased from Charles River (Charles River Italia, Milano, Italy). CD45.1.sup.+ C57BL/6, DO11.10 and 16.2β Transgenic (Tg) (BALB/c background) (25) mice were bred in the Institute specific pathogen free facility. TS/A and TS/A-LACK mouse mammary adenocarcinoma were previously described (17, 19, 26). Exponentially growing 4×10.sup.5 tumor cells were subcutaneously injected in 100.sub.11.1 of PBS in the right flank of syngeneic mice (BALB/c). Typically, five mice per group were used in each experiment.
(49) T Cell Primary Cultures
(50) Twenty days after tumor cell injection mice were sacrificed and the axillary, brachial and inguinal peripheral lymph nodes (LN) draining and distal (non draining LN) to the site of tumor growth were recovered. In the experiments of dendritic cell (DC) vaccination, mice were sacrificed fourteen days after DC administration and the axillary, brachial and inguinal LN were surgically excised. LN cells were cultured in 24 well plates at the density of 5×10.sup.6 in complete medium in the absence or in the presence of recombinant murine IL-7 (200 ng/ml), IL-2 (20 ng/ml), IL-6 (45 ng/ml), or IL-15 (100 ng/ml) (all from Peprotech). In parallel cultures, cells were in vitro stimulated with the LACK-derived MHC II-restricted peptide (5 (25)) and 5×10.sup.6 irradiated syngeneic splenocytes. As a control similar cultures were set up from syngeneic naïve mice. In some experiments, LN cells were labeled with the fluorescent dye CFSE (5-(and-6)-carboxyfluorescein diacetate, succinimilyl ester) at the final concentration of 1 μM accordingly to manufacturer instruction.
(51) Dendritic Cell (DC) Preparation
(52) Bone Marrow derived DC were obtained as previously described (18). Briefly, CD45.2.sup.+ C57BL/6 bone marrow precursors were propagated for 7 days in complete Iscove's medium containing 25 ng/ml recombinant murine GM-CSF and 5 ng/ml recombinant murine IL-4 (Pharmingen, San Diego, Calif.). Then, BMDC were matured at 37° C. in the presence of LPS (1 μg/ml, Sigma, Milan, Italy) for 8 hours and pulsed for 1 hour with 10 μg/ml of the large T Ag-derived Tag IV peptide (18). DC maturation and purity were routinely evaluated by flow cytometry after staining with mAb recognizing CD11c, MHC class II, B7.1, B7.2 and CD40 molecules (all from Pharmingen). 2×10.sup.5 pulsed mature DC were subcutaneously injected in 200 μl of PBS in the right flank of syngeneic C57BL/6 mice.
(53) Flow Cytometry Analysis
(54) I-A.sup.d/LACK multimer staining was previously described. Briefly I-A.sup.d/LACK dimers (MHC II-peptide complexes, 3 μg/sample) are multimerized by the addition of Alexa 488-coupled protein A (Molecular Probes Inc., Eugene, 0.3 μg/sample) in PBS for 30 minutes at room temperature. Free protein A binding sites were saturated by the addition of total IgG (1 μg/sample). 6×10.sup.5 cells were first incubated with a blocking buffer (5% rat serum+95% culture supernatant of 2.4G2 anti-FcR mAb-producing hybridoma cells, 20 minutes) and then stained with the multimers (1 h at 4° C., in PBS supplemented with 0.5% BSA). The cells were then stained with anti-CD4, anti-CD44, anti-CD11b, anti-B220, anti-CD8a mAbs (PharMingen, San Diego, Calif., USA) and TO-PRO-3 (1 nM, Molecular Probes). 3×10.sup.5 CD4.sup.+ or 10.sup.3 CD4.sup.+ I-A.sup.d/LACK.sup.+ events were collected by excluding all of the anti-CD11b.sup.+, anti-B220.sup.+, anti-CD8a.sup.+ and TO-PRO-3.sup.+ events. Where indicated the cells were surface stained with anti-CD4 or anti-CD8 mAb, and anti-CD44, anti-CD127, anti-CD25, anti-CD132 and anti-CD62L mAbs (all from PharMingen except anti-CD127 Ab, A7R34 clone, from Bioscience), and fixed, permeabilized and further stained with anti-Bcl-2 mAb according to to manufacturer instruction.
(55) LACK-Specific Artificial Antigen Presenting Cells (aAPC) and Cytokine Secretion Assays
(56) 5 μm polystyrene sulfate latex beads (Interfacial Dynamics) were coated with I-A.sup.d/LACK dimers (20 μg/ml) and anti-CD28 mAb (37.51; 2 μg/ml) (LACK aAPC) or with anti-CD28 mAb only (control aAPC). Coating of the proteins was monitored by flow cytometry analysis. Typically 5×10.sup.5 LN cells were cultured with 5×10.sup.6 aAPC for 5 hours at 37° C. Brefeldin A (5 μg/ml, Sigma) was added to the cultures for the last 2 hours. Cytokine release induced by LACK aAPC was comparable to the one induced by LACK-pulsed syngeneic splenocytes (not shown). In the case of AH-1 and Tag IV-induced cytokine production, splenocytes were derived from DO11.10 and CD45.1.sup.+ C57BL/6 mice and used as antigen presenting cells. Splenocytes (3×10.sup.7 cells/ml) were pulsed with 1 μM AH-1 (19) and 10 μg/ml Tag IV (18) peptides for 1 hour at 37° C. and then used to stimulate syngeneic LN cells derived from the LN of tumor-bearing mice and DC-vaccinated mice, respectively. Thereafter the cells were stained with anti-CD8, and IL-2 and IFN-γ as described above and with the anti-clonotipic KJ 1.26 mAb, and the anti-CD45.1.sup.+ marker to exclude T cells of APC origin. CD4.sup.+, KJ 1.26″ or CD8.sup.+ CD45.1″ events were then collected on a FACS Calibur. The total number of Ag-specific IL-2.sup.+/IFN-γ.sup.+ T cells was determined by multiplying the percentage by the total number of Trypan Blue-negative LN cells.
(57) Human T Cell Cultures and ELISPOT Assay.
(58) Peripheral blood mononuclear cells (PBMC) were obtained from patients with tuberculosis (TB) and healthy donors by blood centrifugation over Fycoll-Hypaque density gradient and analyzed right away or frozen for future analysis. Where indicated cells were stained with CFSE (1 μM). Cells were cultured in the absence or in the presence of human IL-7 (200 ng/ml), IL-2 (20 ng/ml, IL-15 (100 ng/ml) or IL-6 (45 ng/ml) for 7 days. Where indicated Ciclosporin A (CsA) (0.5 μg/ml) or anti-LFA-1 (5 μg/ml) blocking antibody were added. Cells were then harvested and stained with CD4, CD8, CD3, CD56, CD45RA, CD62L mAbs (all from PharMingen) and analyzed by flow cytometry.
(59) The ELISPOT assay for IFN-γ secretion was performed as previously reported (28). Briefly cells were seeded in duplicate at 5×10.sup.4 cells/well in 96-well plates (MAIPS4510; Millipore, Bedford, Mass.) pre-coated with anti-IFN-γ capture mAb (B-B1; Diaclone, Besancon, France) in the presence of autologous irradiated PBMC (5×10.sup.4 cells/well), and a pool of MTP peptides for 18 h at 37° C. in air plus 5% CO.sub.2. Biotinylated anti-IFN-γ detection mAb (B-G1; Diaclone) was added for 4 h, followed by the addition of streptavidin-alkaline phosphatase conjugate (Amersham Pharmacia Biotech Europe GmbH, Freiburg, Germany) for 1 h. After a washing step, the nitroblue tetrazolium-BCIP (5-bromo-4-chloro-3-indolylphosphate; Sigma, St. Louis, Mo.) chromogenic substrate was added. Individual spot forming cells (SFC) were counted using an automated image analysis system ELISPOT reader (AID-GmbH, Strassberg, Germany). A pool of six synthetic Mycobacterium tuberculosis peptides (MTP; Primm srl, Milano, Italy) with a length of 20 amino acids, >70% purified, derived from the sequences of ESAT-6 and CFP-10 secretory proteins of M. tuberculosis were used at a final concentration of 2 μg/ml per peptide for the detection of a specific response (28). PBMCs in medium alone or stimulated with phytohemagglutinin (PHA-P; Sigma) 5 μg/ml were respectively used as negative and positive controls.
(60) In some instances, MTP-specific IFN-γ release was analyzed at the single cell level by intracellular cytokine secretion assay, Briefly, 0.6×10.sup.6 CFSE labeled cells—were re-stimulated for 6 hours in the presence of human anti-CD28 stimulating mAb (2 μg/ml) and 3×10.sup.6 autologous irradiated (5000 rad) PBMCs pulsed with HLA-DR-restricted MTP (4 μg/ml) or left unpulsed, in negative controls. In the last 5 hours Brefeldin A (10 μg/ml) was added to the cells. Thereafter the cells were fixed, permeabilized and stained with anti-CD4, anti-IL-2 and anti-IFN-γ mAbs and analyzed on a FACS Calibur.
(61) Classification of TB Patients.
(62) Five HIV-seronegative patients with active TB (clinic and culture confirmed) were recovered at the Clinic of Infectious Diseases, S. Raffaele Hospital. They underwent tuberculin skin test (TST) administered by the Mantoux method with 0.1 ml (5 tuberculin units) of Biocinetest-PPD tuberculin (Chiron Italia srl, Milano, Italy). The size of induration was evaluated after 48-72 hours (an induration 10 mm was classified as positive). Peripheral blood was withdrawn before starting any therapy and with a previous written informed consent. Healthy controls (n=8) were selected among HIV-seronegative individuals with no history of TB exposure, no infection and with negative reaction to the TST.
(63) Activation, Culture and Retroviral Transduction of Human T-Cells
(64) Peripheral blood mononuclear cells (PBMC) were isolated by Lymphoprep gradient separation from buffy coats of healthy donors obtained after informed consent (Fresenius, Oslo, Norway). PBMC were cultured in RPMI1640 medium (GIBCO-BRL, Gaithersburg, Md.) supplemented with antibiotics, glutamine and with 10% heat-inactivated FBS (BioWhittaker-Italia, Milano, Italy). PBMC were seeded in 6-well plates (1×10.sup.6/ml) and activated with anti-aCD3 (OKT3 30 ng/ml, OrthoBiotech, Raritan, N.J.) or para-magnetic baCD3/CD28 (3:1 beads/T-cell) (Dynabeads, Dynal Biotech, or Xcyte Therapies Inc., Seattle Wash., USA, Invitrogen). T-cells were enriched by baCD3/CD28 before culture. Cells activated with aCD3 were cultured with human recombinant IL-2 at 600 IU/ml (Chiron, Emeryville, Calif.).
(65) Cells activated with baCD3/CD28 were cultured: 1. In the absence of cytokines; 2. with human recombinant IL-7 at the minimal concentration of 5 ng/ml (Peprotech, London, UK); 3. with human recombinant IL-7 and IL-15 both at the minimal concentration of 5 ng/ml each (Peprotech, London, UK). At day 2 and 3, cells were transduced with the SFCMM3 (39-40) retroviral supernatant by spinoculation at 2400 rpm for 2 h at 37° C. with 8 μg/ml polybrene (Sigma, St Louis, Mo.). The SFCMM3 retroviral vector encodes for the TK suicide gene under the LTR promoter and for a truncated form of the low affinity receptor for nerve growth factor (ΔLNGFR) under the SV40 promoter (27). The retroviral supernatant was provided by Molmed s.p.a. At day 6 after T-cell activation, cell-sized beads used for the stimulation were removed from T-cells, according to manufactureur's instructions. In some experiments, at day 7 after T-cell activation, transduced lymphocytes were positively selected according to the protocol that follows (protocol entitled: Positive immuneselection of transduced lymphocytes).
(66) Cells were cultured up to 14 days. At day 14, fold expansion was calculated by multiplying the percentages of LNGFR.sup.+ cells determined by flow-cytometry with the trypan blue counts. Medium and cytokines were replaced, according to the initial protocol, every 3-4 days. At selected time-points, fold expansion was calculated by multiplying the percentages of ΔLNGFR.sup.+ cells determined by flow-cytometry with the trypan blue counts.
(67) Positive Immuneselection of Transduced Lymphocytes
(68) At day 7 after T-cell activation transduced lymphocytes were positively selected. Since transduced cells expressed ΔLNGFr on their surface, anti-LNGFr antibodies covered with magnetic beads were used to separate transduced from untransduced lymphocytes.
(69) Cells were collected from plates in tubes, washed (1500 rpm, 10 min. at room temperature-RT) and resuspended in WB at a final concentration of 5×10.sup.6/ml. Anti-LNGFr antibody 20.1 was then added to the cell suspension (1 □g/20×10.sup.6) and cells were placed to rotate at 10 rpm for 30 min at RT. T-cells were then washed once and resuspended in WB at 25×10.sup.6/ml. Dynabeads M-450 Sheep anti-Mouse IgG were then added (5×10.sup.6 beads/10.sup.6 positive cells) and cells were placed to rotate at 10 rpm for 30′ at room temperature. Transduced cells were then magnetically selected. To this purpose, tubes were placed near the magnet for 3 min and the negative fraction was discarded. This procedure was repeated for a total of three times. Finally the fraction of cells bound to the beads was removed from the magnet, washed, and resuspended in fresh medium with the appropriate cytokine cocktail at a concentration of 1×10.sup.6 cells/ml.
(70) Flow Cytometry of Transduced T-Cells
(71) Flow cytometry was used for analysis of surface phenotype, transduction efficiency, cell cycle, and cytokine production. The following antibodies (Pharmingen) were used: FITC-conjugated mAb to human CD4, CD8, CD45RA, CD27 and IFN-γ, (PE)-conjugated mAb to human CD4, CD8, LNGFR, CD62L, CD28 and IL-4, peridinin chlorophyll-a protein (PerCP)-conjugated mAb to mouse CD45 (Ly5.1), and allophycocyanin (APC)-conjugated mAb to human CD3 Samples were run through a Facscalibur flow-cytometer (Becton Dickinson, Mountain View, Calif.) after isotype-matched fluorochrome-conjugated irrelevant mAb-stained control and data were analyzed using CellQuest Software (Becton Dickinson). Fluorochrome-conjugated antibodies to CD127, CD122, CCR7, and to mouse CD45 (Pharmingen, San Diego, Calif., USA) were also utilized to stain lymphocytes.
(72) Cytokine Production
(73) For determination of cytokine production, cells were seeded in 24-well plates (1×10.sup.6/ml) and stimulated with 50 ng/ml PMA (Sigma) and 1 μg/ml ionomycin (Sigma). After 4 h, brefeldin A (Sigma) were added for additional 2 h (10 μg/ml). Cells were then stained with the appropriate fluorochrome-conjugated anti-surface marker antibodies and fixed with 1% para-formaldehyde at 4° C. for 10 min. Intracellular staining was performed with the appropriate fluorochrome-conjugated anti-cytokine antibodies after incubation for 20 min at RT in PBS 2% FBS containing 0.05% saponin (Sigma).
(74) CFSE Dilution Assay and Mixed Lymphocyte Reaction (MLR)
(75) Analysis of T-cell alloreactivity was performed at day 10 after initial culture of lymphocytes. Transduced T-cells were stained with CFSE at day 10, and then cultured with irradiated allogeneic PBMCs. CFSE consists of a fluorescein molecule containing a succinimidyl ester functional group and two acetate moieties. CFSE diffuses freely into cells and intracellular esterases cleave the acetate groups converting it to a fluorescent, membrane impermeant dye. The dye is not transferred to adjacent cells. CFSE is retained by the cell in the cytoplasm and does not adversely affect cellular function. During each round of cell division, the relative intensity of the dye decreases by half.
(76) CFSE Staining Procedure:
(77) Cells were washed twice in PBS (in the absence of serum) and adjusted to 2×10.sup.7/ml. CFSE was diluted to 1 μM in PBS and mixed with the cell suspension at a 1:1 ratio. Cells were vortexed quickly, and mixed for 8 min. at RT. FBS was then added at a 1:1 ratio and 1 minute later cells were centrifuged at 2000 rpm for 2 min. Supernatant was then discarded, cells washed twice with a 10% FBS containing solution (PBS or medium).
(78) MLR
(79) At the end of the procedure, CFSE-stained transduced T-cells (responders) were placed in culture in 24-well-plates with 2000 cGy irradiated allogeneic PBMCs (stimulators) in a 1:1 ratio. No cytokines were added to cell culture. CFSE-stained transduced T-cells placed in culture in the absence of stimulators were used as negative control. Cells placed in culture with soluble anti-CD3 antibody were used as positive control.
(80) Read-Outs
(81) At selected time points after stimulation, cells samples were collected and stained with fluorochome-conjugated anti-surface markers monoclonal antibodies. Immediately before FACS acquisition, 1 μl To-Pro-3 solution was added to each FACS sample. To-Pro-3 is a high red intercalating DNA dye, detectable in fluorescence 4, which offers the possibility to co-stain cells with FITC, PE and APC conjugated antibodies. Its function is to reveal the dead cell fraction.
(82) In Vivo Analyses
(83) Mice with immunological defects in the adaptive (scid, recombination-activating genes.sup.−/−) as well as in the innate compartment (NOD, common γ chain.sup.−/−) are commonly used to study human lymphocyte biology in vivo. We utilized NOD/scid mice to test the activity of central memory genetically modified lymphocytes in vivo. Six- to 8-week-old female NOD/scid mice were obtained from Charles-River Italia (Calco, Italy). The experimental protocol was approved by the internal committee for animal studies of our Institute (Institutional Animal Care and Use Committee [IACUC]). Mice were treated according to the following protocols:
(84) Xenogenic Graft-Versus-Host Disease Model
(85) 6-8 weeks old female NOD/scid mice were obtained from Charles-River Italia (Calco, Italy). One week before infusion, mice were transferred from laminar-flow isolators to normal cages and kept under specific pathogen-free conditions receiving sterile water and irradiated pellets ad libitum. The day before the experiment, mice were given 1 mg blocking anti-mouse IL-2Rβ monoclonal antibody i.p. to neutralize residual NK activity. The anti-IL2Rβ antibody was produced as described from the TMβ-1 hybridoma kindly provided by Prof. Tanaka (Osaka University, Japan). At day 0, mice received total body irradiation with a single dose of 350 cGy (gamma irradiation from a linear accelerator) and were immediately infused with unmodified PBL or human lymphocytes transduced with the SFCMM3 retroviral vector (28). Unmodified PBL were obtained from PBMC after the depletion of contaminating monocytes, B- and NK-cells with Pan T-cell isolation kit (Miltenyi, Bergisch Gladbach, Germany). Cells were re-suspended in 500 μl X-VIVO15 medium and infused i.p. Mice were then monitored for GvHD by calculating weight loss. Moribund mice were sacrificed for ethical reasons. Human chimerism was determined weekly by flow-cytometry after bleeding from the tail vein. Human chimerism was calculated as follows: human chimerism (%)=[huCD3.sup.+/(huCD3.sup.++mCD45.sup.+)]×100.
(86) Analysis of Xenogenic GvHD
(87) At week 1, 2 and 3 after T-cell infusion, mice were weighted and evaluated for xenogeneic GvHD according to the following score: weight loss (0 for weight loss <10%, 1 for 10%-25%, 2 for >25%), hunching (0-2), activity (0-2), fur texture (0-2), and skin integrity (0-2), maximum index 10. Weight loss was also estimated as an independent variable, since it was considered the most objective criterion (Table 1).
(88) TABLE-US-00001 TABLE 1 Assessment of clinical xeno-GvHD in human T-cells infused mice. Criteria Grade 0 Grade 1 Grade 2 Weight loss <10% 10-25% >25% Posture Normal Hunching noted Severe hunching impairs only at rest movement Activity Normal Mild to moderately Stationary unless decreased stimulated Fur texture Normal Mild to moderately Severe ruffling/poor ruffling grooming Skin integrity Normal Scaling of paws/tail Obvious areas of denuded skin
Allogeneic GvHD Model
(89) In the allogeneic GvHD model, mice were transplanted with human skin and infused with allogeneic genetically modified lymphocytes to evaluate their ability to home to the human skin and mediate an allogenic GvH reaction. One week before transplantation, mice were transferred from laminar-flow isolators to normal cages and kept under specific pathogen-free conditions receiving sterile water and irradiated pellets ad libitum. Around three weeks before human T-cell infusion, mice are anesthetized with 12-18 mg avertin/mouse intraperitoneally. They were then depilated on the back, and an horizontal skin incision was performed bilaterally on the animal's back. A subcutaneous pocket was then opened, and a small piece of human abdominal epidermis (deprived from dermal fat and connective tissue) was introduced. At the end of the procedure, the wound was sutured. Since mice temperature progressively decreases during the operation, animals were placed in a heated box for about 30 min. and finally transferred into their cages.
(90) Human T-Cell Infusion
(91) To facilitate engraftment of human lymphocytes in NOD/scid mice, we functionally inactivated NK cells with anti-mouse IL-2 receptor β (TMβ-1) antibodies prior to lymphocytes transfer. The antibody was produced from the TMβ-1 hybridoma kindly provided by Prof Tanaka (Osaka University, Japan). At day 0, mice received total body irradiation with a single dose of 300 cGy (γ irradiation from a linear accelerator). Animals were then weighted and immediately infused with transduced human lymphocytes that had been harvested at day 9 after initial stimulation. Cells were infused intravenously in 250 μL saline solution.
(92) Analysis of T Cell Engraftment
(93) At week 1, 2 and 3 after T-cell infusion, about 300 μl blood/mouse was harvested from a little incision in the tail vein and collected in heparin-containing tubes. Red blood cells were lysed with a 3 min. exposure to ACK and then stained for cytofluorimetric analysis as described in the paragraph entitled “Staining for surface markers and cytofluorimentric analysis”.
(94) Analysis of Allogeneic GvHD
(95) At week 3 after T-cell infusion, or earlier in case of severe GvHD, mice were sacrificed and the two pieces of human skin removed bilaterally. Formalin-fixed, paraffin-embedded skin was cut in 4-μm thick sections and stained with hematoxylin and eosin for morphologic evaluation. Immunohistochemical assessment for the presence of human T lymphocytes was carried out with monoclonal anti-human CD3 antibody (Dako, Glostrup, Denmark) at 1:100 dilution, by way of the avidin/biotin peroxidase complex method using an automized Dako immunostainer. Staining reaction was revealed by the tetrahydrochloride chromogen method and sections were counterstained with hematoxylin. Pictures were taken with a Zeiss Axiocam HRC.
(96) Statistical Analyses
(97) For each variable considered in this study, mean, median and standard devation were calculated. All statistical analyses were performed by using Microsoft Excel 2003 (Microsoft, Redmond, Wash.) and its add-in form Statcel2 (OMS publish, Saitama, Japan). Scheffe's F test following analysis of variance (ANOVA) was performed for parametric data, and Mann-Whitney's U test following Kruskal-Wallis test was performed for non-parametric data.
(98) Results and Discussion
(99) IL-7 Favors the Detection of Rare Tumor-Specific CD4.sup.+ T Cells without the Need of Ag-Driven Cell Expansion.
(100) The enumeration of Ag-specific T cells might be critical to several clinical conditions, for which the presence of Ag-specific T cells, is of diagnostic and prognostic interest. The authors recently developed a preclinical mouse model of tumor-disease with TS/A tumor cells expressing the Leishmania Major-derived model Ag LACK (TS/A-LACK). While LACK-specific naïve CD4.sup.+ T cells can not be identified in unmanipulated BALB/c mice (29), the authors recently identified LACK-specific Ag-experienced CD44.sup.high CD4.sup.+ T lymphocytes in TS/A-LACK tumor-bearing mice by fluorescent MHC class II/Ag multimers staining and by LACK-specific IL-2 and IFN-γ intracellular release (20).
(101) In the effort of improving the detection and cloning of Ag-specific T cells, the authors investigated whether IL-7, known to favor survival of memory CD4.sup.+ T cells (7, 9-14) might enrich the frequency of tumor-specific CD4.sup.+ T cells. Control TS/A and TS/A-LACK tumor cells were subcutaneously injected in BALB/c mice. Twenty days after tumor cell injection, all the mice had developed measurable tumors. Mice were sacrificed and the tumor draining and non draining LN were surgically excised. While the formers contained a population of LACK-specific Ag-experienced CD44.sup.high CD4.sup.+ T lymphocytes capable of IL-2 and IFN-γ production, the latter remained ignorant of the tumor and present LACK-specific naïve CD4.sup.+ T lymphocytes (31). The frequency of LACK-specific CD4.sup.+ T cells was analyzed ex vivo, and after 7 days in culture in the presence of recombinant IL-7 without any further Ag stimulation (
(102) IL-7 and IL-2, but not Antigenic Stimulation Favor the Accumulation of Tumor-Specific CD4.sup.+ T Cells.
(103) Re-stimulation with Ag is most commonly used to expand, and in some instances to identify Ag-specific CD4.sup.+ T cells (34). Furthermore, in addition to IL-7, also IL-2 and IL-15 control memory T cells proliferation (13, 35-37). Hence, LN cells from TS/A-LACK tumor-bearing mice were cultured in the presence of irradiated singeneic APC unpulsed (APC) or pulsed with the LACK-derived peptide (Ag/APC) or in the presence of optimal amounts of IL-7, IL-2, IL-15 and IL-6, as control, and analyzed by flow cytometry. The frequency of CD4.sup.+ I-A.sup.d/LACK.sup.+ CD44.sup.high T cells was slightly higher in cultured cells when compared to the one found ex vivo (
(104) IL-7 and IL-2 Sustain the Ag-Independent Spontaneous Proliferation and Survival of In Vivo-Primed Tumor-Specific CD4.sup.+ T Cells.
(105) The authors next investigated the mechanism by which IL-7 and IL-2 favor the accumulation of in vivo-primed LACK-specific CD4.sup.+ T cells. First the authors analyzed the ability of these cytokines to support the expansion of these cells in vitro. LN cells were labeled with the CFSE vital dye, and cultured for a week in the absence or in the presence of the recombinant cytokines. In the absence of exogenous cytokines LN cells derived from naïve mice did not proliferate and retained their original CFSE content (
(106) In addition to IL-7, also IL-2 supported the in vitro proliferation of a fraction of CD4.sup.+ T cells and increased the number of LACK-specific memory lymphocytes. In contrast, IL-15 and IL-6 failed to support either proliferation of the cells, or the accumulation of LACK-specific CD4.sup.+ T cells over the one found in control (nil) cultures (
(107) Even in this unrelated model, IL-7 and IL-2 enrich LN cultures of Ag-experienced CD4.sup.+ T cells by sustaining their in vitro proliferation bypassing the need of Ag-stimulation.
(108) To further characterize the relative potency of IL-7 and IL-2 in promoting the accumulation of Ag-experienced CD4.sup.+ T cells, CFSE labeled cells were stained with the fluorescent dye TO-PRO-3, able to identify viable and dead cells within proliferating cells. IL-7 best preserved the viability of the cultures with only 15% of TO-PRO-3.sup.+, dead cells after a week. At difference, up to 47% of the cells maintained in IL-2 and 60%, 57%, and 73% of the cells cultured in the absence of exogenous cytokine or in the presence of IL-15 and IL-6 resulted TO-PRO-3.sup.+ (
(109) The ability of IL-7 and IL-2 to favor T cell survival is linked to their capacity to regulate the expression of the anti-apoptotic factor Bcl-2 (10, 38). Thus, the authors analyzed Bcl-2 levels in CFSE-labeled LN cultures maintained in the absence and in the presence of recombinant cytokines. In every culture conditions, with the exception of IL-15, CFSE dim cells expressed optimal levels of Bcl-2 (
(110) IL-7, IL-2 and IL-15 Expand Ag-Specific Specific Memory CD8.sup.+ T Cells in an Ag-Independent Manner.
(111) To further address the general usefulness of the Ag-independent short-term culture in IL-7 to reveal in vivo generated Ag-specific T cells the authors investigated whether Ag-specific CD8.sup.+ T lymphocytes could be traced in a different context from tumor disease. The major aims were: 1) to evaluate the applicability of the invention for the tracing of in vivo Ag-experienced CD8.sup.+ T cells, and not only in vivo Ag-experienced CD4.sup.+ T cells, 2) to evaluate the applicability of the invention in a clinical setting (active vaccination), different from the diagnosis of anti-tumor immune responses.
(112) In an attempt to investigate whether IL-7 could be used to reveal Ag-specific T cells in a clinical setting different from the one of tumor-disease, we analyzed peptide-specific CD8.sup.+ T cells induced by a dendritic cell (DC)-based vaccine. C57BL/6 mice were vaccinated with bone marrow derived DC pulsed with the MHC class I restricted Tag IV peptide (DC-Tag) derived from the SV40 Large T antigen (18). Fourteen days later LN cells were analyzed by Ag-specific intracellular cytokine release ex vivo and after the cytokine-driven cultures. As a control LN cells were also derived from naïve unvaccinated C57BL/6 mice. Ex vivo, Tag IV specific CD8.sup.+ T cells capable of producing only IFN-γ or IL-2 and IFN-γ after Ag re-stimulation were undetectable in naïve mice, and detectable at low frequencies in DC-vaccinated mice (0% and 0.37%, respectively). After 7 days in culture with IL-7, IL-2 and IL-15 in the absence of Ag re-stimulation the frequency (3.94%, 1.83% and 1.95%, respectively) as well as the total number (
(113) In the same cultures the authors analyzed the relative enrichment of Tag IV-specific CD8.sup.+ T cells in comparison to the total CD8.sup.+ T cells (
(114) IL-7 Reveals Antigen-Specific CD8.sup.+ T Cells Otherwise Undetectable Ex Vivo.
(115) TS/A cells naturally express the envelope protein gp70 of an endogenous MuLV for which an immunodominant epitope was previously described (AH-1, (19)). In their further attempt to address if the culture in IL-7 might also aid the identification of rare Ag-specific CD8.sup.+ T cells, the authors compared the AH-1-specific CD8.sup.+ T cell responses ex vivo and after a week in culture without or with IL-7, IL-2, IL-15 and IL-6 in the absence of Ag re-stimulation. Lymphocytes were analyzed by intracellular cytokine release upon stimulation with unpulsed and AH-1-pulsed syngeneic splenocytes (
(116) Interleukin-7 Synergizes with a Cyclosporin A-Sensitive Signal for the Selective Expansion of Memory CD4.sup.+ T Cells.
(117) Proliferation and survival of memory CD4.sup.+ T cells in vivo relies on both IL-7 as well as TCR-driven events (9). In vitro, TCR-driven proliferation of human memory T cells requires intact ERK activity, while cytokine-driven homeostatic cell division relies on p38 and is insensitive to Cyclosporin A (CsA) (13). The authors thus analyzed the requirements for the IL-7-driven accumulation of intermediate memory T cells by culturing lymphocytes in the presence of blocking antibodies or of inhibitors of selected signaling pathways. Cells were derived from the LN of naïve or TS/A-LACK-tumor bearing 16.20 mice, which are transgenic mice having a sizeable frequency of LACK-specific naïve CD4.sup.+ T cells (25), labeled with CFSE and cultured in the presence of optimal IL-7 amounts, and the indicated inhibitory agents. As control, naïve T cells were stimulated with LACK-pulsed antigen presenting cells (APC) in the absence or in the presence of the selected inhibitors (
(118) Together these findings indicate that IL-7 is able to drive the accumulation of fast proliferating IL-2/IFN-γ.sup.+ intermediate memory CD4.sup.+ T cells by the synergy with a cell-derived CsA sensitive signal possibly mediated by adhesion molecules and/or self peptide/MHC interaction.
(119) IL-7 Sustains the Selective Accumulation of Fast-Dividing Peripheral Blood Human CD4 Memory T Lymphocytes.
(120) It was previously reported that IL-7 and IL-15 sustain a slow homeostatic-like cell division of both central memory and effector human memory T cells (13). The authors investigated whether high-density culture conditions and optimal IL-7 amounts could instead reveal a population of fast-dividing intermediate memory T cells among peripheral blood-derived T lymphocytes. To this aim PBMC were derived from healthy donors, labeled with CFSE and cultured for 7 days at different cell densities in the absence or the presence of optimal IL-7 amounts (
(121) In addition to IL-7, also IL-2 and IL-15 sustained the in vitro proliferation of human CD4.sup.+ T cells (
(122) IL-7 Driven T Cell Expansion of Peripheral Blood Human T Lymphocytes is Sensitive to Cyclosporin A.
(123) The IL-7 and IL-15-driven slow homeostatic-like cell division of human memory T cells was reported to be insensitive to CsA, and instead rely on p38-dependent signaling (13).
(124) The authors next investigated the signaling required for the IL-7 driven expansion of the intermediate memory fast-proliferating human T cells. As in the case of mouse cells, the IL-7-driven accumulation of fast-proliferating CD4.sup.+ T cells was sensitive to CsA, and to RAPA, and to a lesser extent to SB (
(125) IL-7-Driven Short-Term Cultures Aid the Enumeration of Mycobacterium tuberculosis-Specific CD4.sup.+ T Cells in Human Subjects.
(126) Having determined that IL-7 sustains the in vitro expansion of a fast-proliferating memory T cells possibly programmed in vivo to proliferate in vitro, the authors investigated whether this could be exploited for the clinical investigation of immune-related pathologies. To this aim, crio-preserved PBMC samples of M. tuberculosis-infected (TB) patients were analyzed at the time of thaw or after a week in culture with optimal IL-7 amounts, by MTP-specific ELISPOT analysis (43). Patients were chosen based on their clinical history and manifestation of acute TB (clinic and culture confirmed), on their positive reaction to the TST, and on the ability to respond to MTP in the ELISPOT-IFN-γ assay. Crio-preserved PBMC from not infected healthy donors were also analyzed as control. Pt #1 showed a sizeable number of IFN-γ.sup.+ spots upon MTP-specific re-stimulation (
(127) IL-7-Driven Short-Term Cultures Aid the Enumeration of Candida Antigen-Specific Human T Lymphocytes.
(128) In addition to MTP-specific T cells, the authors also investigated whether IL-7 could enhance the identification of T lymphocytes specific for Candida Albicans-derived antigens. To this aim, PBMCs from Pt #1 were analyzed at the time of thawing or after a 7 days culture in plain medium or in the presence of IL-7, by an ELISPOT assay performed with unpulsed or C. Albicans-derived Ag-pulsed irradiated autologous PBMC. As in the case of M. Tuberculosis-specific T cell responses, also C. Albicans-specific T cells capable of IFN-γ release were enriched for by the short-term culture in IL-7 (
(129) The Adoptive Cell Therapy with IL-7/IL-15 Cultured Memory T Cells Delays Tumor Growth In Vivo.
(130) Having determined that IL-7 determines the accumulation of in vivo primed memory CD4 T cells, and that IL-15 best drives the expansion of CD8 memory T cells, the authors evaluated whether the expanded populations have a clinical relevance. To address this point lymph nodes were derived from TS/A-LACK tumor-bearing mice and cultured for 7 days in optimal cell density (5×10.sup.6 cells/ml) and optimal cytokines amounts (50 ng/ml). As a control naïve T cells derived from a control mouse were also cultured in the same conditions. Thereafter 10.sup.7 cultured cells bearing comparable frequencies of CD4 and CD8 T cells were adoptively transferred into naïve BALB/c mice. 48 hours later mice were challenged with 300.000 TS/A-LACK cells and tumor growth was monitored overtime. As shown in
(131) Generation of Gene-Modified Central Memory Human T-Cells
(132) Cell proliferation is required for retroviral transduction of T lymphocytes. The authors activated PBMC with aCD3 or baCD3/CD28. Cells were activated with baCD3/CD28 and cultured with IL-7 and IL-15 or with aCD3 and cultured with IL-2 (
(133) Polyclonal Activation Required for Retroviral Transduction of T Lymphocytes Enriches for Memory Cells.
(134) To determine the relative distribution of memory subsets in human T-cells transduced with the retroviral vector, the authors analyzed CD45RA/CD62L co-expression. At day 14, transduced T-cells activated with aCD3 and cultured with IL-2 were mainly CD45RA.sup.− CD62L.sup.−, i.e, effector memory cells. On the contrary, transduced CD4.sup.+ T-cells activated with baCD3/CD28 and cultured IL-7 and IL-15 were highly enriched for CD45RA.sup.−CD62L.sup.+, i.e., central memory cells (
(135) Functional Correlates of Gene-Modified Central Memory Human T-Cells
(136) Central and effector memory human T lymphocytes, as identified by surface phenotype, differ in the ability to produce effector cytokines. At day 14, the authors re-stimulated the two populations of gene-modified T-cells and analyzed them for cytokine production. CD4.sup.+ T-cells stimulated with aCD3 and cultured with IL-2 efficiently produced the prototypical effector cytokine IFN-γ In sharp contrast, the majority of CD4.sup.+ T-cells stimulated with baCD3/CD28, IL-7 and IL-15 were un-polarized cells and neither produced IFN-γ nor IL-4 (
(137) GvHD Potential of Gene-Modified Central Memory Human T-Cells.
(138) Different xenograft models have been proposed to study GvHD induced by human lymphocytes. In order to evaluate the relative anti-host reactivity of the two suicide gene-modified central and effector memory human T lymphocytes in vivo, these populations were infused into NOD/scid mice conditioned with non-lethal irradiation and anti-NK antibodies. Control mice were infused with human purified syngeneic PBL. The authors observed that central memory gene-modified lymphocytes were more efficient at engrafting than their effector memory counterpart (human chimerism at week 1: average 0.45% range 0.2-1.1 for effector memory genetically modified cells vs average 4.5% range 4.1-5.2 for central memory genetically modified cells, Table 2. 5 out of 6 mice infused with effector memory gene-modified T-cells presented a decreased human chimerism after week 1 and did not show GvHD. On the other hand, persistent human chimerism was observed in the majority of mice infused with central memory suicide gene-modified T-cells and 4 mice out of 6 developed severe GvHD.
(139) TABLE-US-00002 TABLE 2 Engraftment and graft-versus-host disease Effector memory Central memory PBL TK.sup.+ cells TK.sup.+ cells Human chimerism in 3.6 (2.5-5) 0.45 (0.2-1.1) 4.5 (4.1-5.2) % (range).sup.a GvHD incidence.sup.b 6/6 1/6 4/6 .sup.aAverage (range) at week 1 after infusion .sup.bDefined as weight loss >10% from initial body weight
Generation of Gene-Modified Central Memory Human T Cells
(140) To determine the minimal requirements to obtain a number of gene-modified central memory human T-cells suitable for clinical application, we compared the following five T-cell transduction conditions: 1. soluble anti-CD3 antibodies (OKT-3)+high doses of interleukin 2 (600 UI/ml); 2. anti CD3/CD28 cell sized beads without any cytokine; 3. anti CD3/CD28 cell sized beads+low doses of IL-2 (200 IU/ml); 4. anti CD3/CD28 cell sized beads+low doses of IL-7 (5 ng/ml); 5. anti CD3/CD28 cell sized beads+low doses of IL-7 (5 ng/ml)+low doses of IL-15 (5 ng/ml).
(141) Cells were transduced and cultured following protocols described in materials and methods. These experiments produced the following results:
(142) 1. Activation with Anti CD3/CD28 Beads Allows Higher Transduction Efficiency than Activation with Soluble Anti-CD3 Antibodies
(143) As shown in
(144) 2. Activation with CD3/CD28 Beads Followed by Culture in the Presence of Cytokine (IL2, IL7+IL15 or IL7) Preserves the Physiological CD4/CD8 Ratio in Transduced T Lymphocytes.
(145) To evaluate the ability of our transduction protocols in preserving the physiologic CD4/CD8 ratio, we analysed the CD4/CD8 ratio of transduced cells produced by different protocols, 10 days after initial stimulation. As shown in
(146) 3. Activation with CD3/CD28 Beads Followed by Culture in the Presence of Cytokine (IL2, IL7+IL15 or IL7) Induces a Significantly Higher Proliferation Rate of Transduced Cells than Other Culture Conditions.
(147) Protocol of ex vivo gene transfer designed for clinical application must fulfil to the relevant criteria related to feasibility: one of the major feasibility issue in the clinical translation of a gene therapy approach relates to cell number and cell expansion in vitro. As shown in
(148) 4. Activation with Anti CD3/CD28 Beads Generates Mainly Central Memory CD8.sup.+ and CD4.sup.+ Transduced Lymphocytes
(149) We investigated the immunophenotype of transduced cells obtained by different culture conditions through FACS staining for CD3, CD4, CD8, CD45RA, and CD62L 10 days after initial stimulation. We observed that a very high fraction (about 80%) of both CD8.sup.+ and CD4.sup.+ T-cells, which had been stimulated with anti CD3/CD28 beads, was CD45RA.sup.− CD62L.sup.+: this pattern corresponds to central memory T-lymphocytes. On the contrary, OKT3-stimulated T-cells showed 60% CD8.sup.+ and 45% CD4.sup.+ effector memory T-cells (CD45RA−CD62L−) versus 30% CD8.sup.+ and 50% CD4.sup.+ central memory TK.sup.+ lymphocytes. (
(150) γ-Chain Receptor Expression During Culture of Transduced Lymphocytes
(151) Cytokine receptors' expressions are tightly regulated during T-cell stimulation. We analysed the expression kinetic of γ-chain cytokine receptors during the different T cell culture and transduction protocols, as a measure of T cell functions and potential. To this purpose, we performed cytofluorimetric analysis after staining transduced cells with fluorchrome-labeled antibodies to CD122 (IL-2/15 receptor common β chain), CD25 (IL-2 receptor α chain) and CD127 (IL-7 receptor α chain) at different time-points after first stimulation.
(152) 1. IL-2/15 Receptor 13 Chain (CD122) Expression does not Change Among the Different Transduction Protocols
(153) During the course of immune responses IL-2/IL-15 receptor β expression increases after T cell activation and then decreases to an intermediate level of expression that is retained throughout the memory-cell phase (13).
(154)
(155) 2. Stimulation with Beads CD3/CD28 Promotes an Intense and Prolonged Expression of IL-2 Receptor α in Transduced T-Cells
(156) IL-2 receptor α (CD25) is a relevant activation marker for T-lymphocytes. In physiological conditions, naïve T cells do not express CD25; however, its expression is rapidly upregulated by T-cell activation and usually declines before the proliferative peak of the response.
(157) In transduced cells activated by beads, independently from subsequent culture conditions, flow cytometry analysis showed that at day 2 after stimulation the majority of T-cells underwent a significant increase in IL-2 receptor α expression. On the contrary, only 40% of transduced cells activated with soluble OKT3 up-regulated the receptor (
(158) 3. Transduced Cells Activated with Anti-CD3/CD28 Beads+IL-7 Show the Maximal Expression of IL-7 Receptor α, a Marker of Long-Surviving Memory T-Cells
(159) In physiological conditions, IL-7 receptor α (CD127) is constitutively expressed by naïve T cells. Its expression is downregulated by T-cell activation (in a specular manner compared to CD25) and such down-regulation might promote cell death. Conversely, expression of CD127 increases as the immune response proceeds, reaching high levels in memory T cells. IL-7 is a potent survival factor for memory T-lymphocytes: triggering of the receptor by IL7 promotes T cell survival and proliferation and protects cells from apoptosis through different intracellular signal pathways. We analysed the kinetic of CD127 expression in our transduced cells. IL-7 receptor α underwent a deep down-regulation after stimulation (approximately between day 1 and day 6). From day 7, its expression progressively increased and interestingly, we observed a significant difference in the proportion of CD127+ transduced cells obtained with beads CD3/CD28 and IL-7 compared to all other conditions (
(160) Transduced Lymphocytes Generated with Beads+IL-7 have the Highest Alloreactive Potential
(161) From the results previously shown, it emerges that activation with anti-CD3/CD28 magnetic beads and the adjuvant effect of cytokines (in particular IL-7) are important factors for the generation of central memory transduced T-lymphocytes, with a high survival potential. Our next aim was to investigate whether these CM transduced T-lymphocytes were actually able to elicit a powerful and effective immune response. We addressed this issue in vitro by stimulating transduced cells with allogeneic antigens and we obtained the following results:
(162) 1. Transduced Lymphocytes Generated with Beads+IL-7 have the Highest Proliferative Potential, when Stimulated with an Allogenic Antigen.
(163) Transduced T cells generated with each of the five conditions were stained with CFSE and co-cultured with irradiated allogeneic PBMCs. After 1 week we counted cell numbers and analyzed CFSE dilution by FACS to evaluate the percentage of dividing cells. As shown in
(164) 2. Transduced Lymphocytes Generated with Beads+IL-7 have the Lowest Sensitivity to Death.
(165) In order to maintain T-lymphocytes homeostasis, massive T-cell activation in response to an allogeneic challenge is usually followed by an extensive apoptotic program, IL-2 being the main player in the so called “activation induced cell death” (AICD). Mechanisms to counteract AICD are required for the development of an efficient and long-lasting immunological memory after primary immune responses. To investigate the sensitivity of transduced cells to AICD, we stained allo-stimulated CFSE.sup.+ lymphocytes with To-Pro 3, a fluorescent dye, which selectively binds to dead cells. We calculated the number of dead cells in dividing and non dividing transduced cell populations, in order to evaluate respectively activation induced cell death and death by neglect (
(166) In accordance with this observation, transduced lymphocytes generated with beads CD3/CD28 and IL-2, who proved highly sensitive to cell death, showed the lowest proportion of cells expressing CD127.sup.+ cells (30%). FACS plots in
(167) 3. Transduced Lymphocytes Generated with Beads+IL-7 Preserve a Central Memory Phenotype after Allogeneic Stimulation.
(168) Immunological memory is ensured by a self-renewal capacity of memory cells, that, upon antigen re-enconteer, divide, and generate both effectors, able to directly eliminate the pathogen, and memory cells, able to protect the host long-term. To verify whether central memory genetically modified lymphocytes had this self-renewal capacity, we analysed the expression of CCR7 (a marker of central memory cells) on CFSE-stained cells, at day 10 after allogeneic stimulation. As shown in
(169) Transduced Lymphocytes Generated with Beads+IL-7 have the Highest Alloreactive Potential In Vivo
(170) To evaluate the efficacy of central memory transduced T-cells in vivo, we established a new chimeric model, based on NOD/Scid mice transplanted with human skin. Based on results obtained in vitro we decided to investigate the potency of genetically modified central memory lymphocytes generated with beads CD3/CD28+IL-7, beads CD3/CD28+IL-2 and to compare the functional activity of these cells with genetically modified effector memory lymphocytes, generated with OKT-3 and IL-2, and currently utilized in clinical trials. After infusion of transduced cells, xenogeneic T-cell reactivity was determined by clinical observation, while allogeneic GvHD was evaluated histologically and correlated to the analysis of human skin infiltration by transduced cells. Results of these studies are summarized here below:
(171) Genetically Modified Cells Generated with CD3/CD28 and IL-7 Rapidly Engraft in Skin-Transplanted NOD/Scid Mice
(172) The number of human T-lymphocytes in mice peripheral blood increased from week one to week 2 after infusion. Transduced cells generated with beads engrafted at higher extent than transduced cells generated with OKT-3. The difference was more evident at the second week after T cell infusion (
(173) Activation with Beads CD3/CD28 and IL-7 Stimulation Confer to Transduced Cells the Highest Reactivity Against Xenogeneic Antigens.
(174) Xenogeneic GvHD was monitored according to a clinical score described in material and methods and by measuring weight loss. Infused NOD/Scid mice progressively lost their weight and some of them eventually died for xenogeneic GvHD or were sacrificed for ethical reasons. The most xeno-reactive T-cells were those stimulated with anti CD3/CD28 beads and cultured with IL-7, followed by transduced cells generated with CD3/CD28 beads and IL-2. The stimulation with soluble anti CD3 antibodies did not generate strongly xeno-reactive T-cells, as mice infused with these lymphocytes did show neither a significant body weight loss, nor the appearance of other clinical xeno-GvHD signs (
(175) Activation with Beads CD3/CD28 and IL-7 Stimulation Confer to Transduced Cells the Highest Reactivity Against Allogeneic Antigens
(176) Our NOD/Scid human skin chimera mouse model consists of NOD/Scid mice, that had undergone skin transplantation, through the insertion of two pieces of human abdominal epidermis into two subcutaneous pockets on the mouse back and that were subsequently infused intravenously with genetically modified cells. Human skin transplantation allows to investigate T-cell reactivity against allogeneic antigens, by histological studies. Transplanted human skin, indeed, contains not only epidermal and stromal cells, but also some antigen presenting cells, which are able to attract circulating lymphocytes and possibly promote their activation. Three weeks after human T-cell infusion, we sacrificed NOD/Scid chimera mice and removed human skin bilaterally. All biopsies were subsequently blindly analysed by pathologists through both ematossilin-eosin (EE) and anti human CD3 staining. We observed a massive human T-cell infiltration in the context of human skin, only in mice that had been infused with “beads+IL-7” stimulated T-lymphocytes. In “OKT-3” conditions the level of T-cell tissue infiltration, if any, was clearly spare.
CONCLUSION
(177) Although it is generally recognized that T cells play a central role in the generation of immunity to pathogens, to tumors, and to immuno-deficiencies and in autoimmune disorders, it has been difficult to use them as diagnostic and prognostic markers of immunocompetence in humans. Furthermore, techniques suitable for the expansion of non polarized poly-functional intermediate and central memory lymphocytes are currently missing.
(178) The results described in this report of invention identify new culture conditions which:
(179) A) allow the Ag-independent accumulation of in vivo primed memory T cells;
(180) B) guide expansion of central memory lymphocytes in spite of lymphocyte polyclonal stimulation and genetic manipulation.
(181) Results indicate that IL-7, and IL-15 can be used to enrich biological samples, such as peripheral LN, blood, and tumor for in vivo primed Ag (tumor/pathogens/allergens/self antigens)-specific CD4.sup.+ or CD8.sup.+ T cells. When compared to IL-2, IL-7 better maintained the original lymphocyte phenotype and representation and better favored the survival of all T lymphocyte subsets, allowing the detection and expansion of rare CD4.sup.+ T memory lymphocytes.
(182) The ability to enumerate Ag-specific CD4.sup.+ or CD8.sup.+ T cells in the context of chronic viral infection, autoimmune disease, vaccination or immunotherapy would provide a direct measure for the patient immunocompetence or disease and assist clinicians in the choice of the most appropriate therapy. Furthermore, the possibility to enrich in vivo-primed memory T cells without altering their phenotype might improve their characterization, as well as their exploitation for the immune response in adoptive immunotherapy strategies. Finally, genetically modified central memory T-cells can be obtained upon CD3/CD28 activation and culture with homeostatic cytokines. When infused in conditioned immunodeficient hosts genetically modified central memory T-cells i) engraft and expand at significantly higher levels than effector memory genetically modified T cells and ii) are more potent than effector memory genetically modified lymphocytes at inducing an immune response to host and allogeneic antigens.
(183) These results demonstrate that fully functional central memory gene-modified lymphocytes can be obtained and exploited for the cure of human diseases.
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