METHOD OF AMPLIFYING A POPULATION OF ANTIGEN-SPECIFIC MEMORY CD4+ T CELLS USING ARTIFICIAL PRESENTING CELLS EXPRESSING HLA CLASS II MOLECULES

Abstract

The present invention relates to method of amplifying a population of antigen-specific memory CD4+ T cells using artificial presenting cells expressing HLA class II molecules. In particular, the present invention relates to a method of amplifying a population of antigen-specific memory CD4+ T cells comprising the steps of i) providing a population of artificial antigen presenting cells consisting host cells that are genetically modified to stably express at least one MHC class II molecule along with at least one accessory molecule ii) loading the population of artificial antigen presenting cells of step i) with an amount of at least one antigen of interest and iii) coculturing the suitable population of a T cells with the population of artificial antigen presenting cells of step ii).

Claims

1. A method of amplifying a population of antigen-specific memory CD4+ T cells comprising the steps of i) providing a population of artificial antigen presenting cells consisting host cells that are genetically modified to stably express at least one MHC class II molecule along with at least one accessory molecule ii) loading the population of artificial antigen presenting cells of step i) with an amount of at least one antigen of interest iii) coculturing the suitable population of a T cells with the population of artificial antigen presenting cells of step ii).

2. The method of claim 1 wherein the host cell is deriving from the hematopoietic lineage and is selected from the group consisting of human, murine, rodentia, insect, and any other mammalian cells.

3. The method of claim 1 wherein the host cell is a murine cell.

4. The method of claim 1 wherein the MHC class II molecule is selected from the group consisting of HLA-DQ molecules, HLA-DP molecules and HLA-DR molecules.

5. The method of claim 4 wherein the MHC class II molecule is selected from the group consisting of HLA-DR1, HLA-DR15, HLA-DR51 and HLA-DR11 molecules.

6. The method of claim 1 wherein the host cell is a fibroblast, and more particularly a murine fibroblast such as a NIH/3T3 mouse fibroblast.

7. The method of claim 1 wherein the accessory molecule is selected from the group consisting of co-stimulatory molecules and adhesion molecules.

8. The method of claim 7 wherein the co-stimulatory molecule is CD80.

9. The method of claim 7 wherein the adhesion molecule is CD54 and/or CD58.

10. The method of claim 1 wherein the host cell is genetically modified to stably express the CD80, CD54 and CD58 molecules.

11. The method of claim 1 wherein the antigen is a peptide or a whole protein.

12. The method of claim 1 wherein the antigen is a viral antigen, a bacterial antigen a fungal antigen or a protozoal antigen.

13. The method of claim 1 wherein the antigen is a tumor-associated antigen, an auto-antigen, or an allergen.

14. The method of claim 1 wherein the antigen is a molecule that is exogenously administered for therapeutic or other purposes and may trigger an unwanted immune response.

15. The method of claim 1 wherein the population of CD4+ T cells is a population of CD4+ T cells generated after primary stimulation of total PBMCs with the antigen of interest.

16. The population of antigen-specific memory CD4+ T cells amplified by the method of claim 1.

17. A method of treating a cancer, an infectious disease, an autoimmune disease, an allergy, an immune reaction against a molecule that is exogenously administered for therapeutic or an immune reaction against a grafted tissue or grafted hematopoietic cells or grafted blood cells in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the population of the population of antigen-specific memory CD4+ T cells of claim 16.

Description

FIGURES

[0061] FIG. 1: Peptide presentation by AAPCs.

[0062] AAPCs or B-EBV cell lines have been loaded with different concentrations of FVIII (A), HA (B), MBP (C) or control peptide and used to stimulate FVIII, HA or MBP specific CD4+ T cell clones. For FVIII or HA peptide, frequencies of activated T cells were evaluated by intracellular IFN-? staining (ICS) and FACS analysis. Representative results are shown on graphics, and represent percentages and MFI of IFN-?+ cells among CD4+ T lymphocytes. Proliferation of the MBP specific T cell clone was measured by incorporation of 3H-TdR and results expressed in cpm.

[0063] FIG. 2: Protein presentation by AAPCs.

[0064] Kinetics analysis of specific CD4+ T cell clone stimulation with AAPCs or B-EBV cell lines loaded with 40 nM of FVIII (A) or HA (B) proteins. At optimal time, AAPCs or B-EBV cell lines incubated with different concentrations of FVIII protein (12 h) or HA protein (6 h) were used to stimulate specific CD4+ T cell clones. Frequencies of activated T cells were evaluated by ICS and FACS analysis. Dose-response results are shown as percentages and MFI of IFN-?+ cells among CD4+ T lymphocytes.

[0065] FIG. 3: Evaluation of presentation mechanisms by AAPCs.

[0066] Ag containing medium or supernatant from overnight incubation of AAPCs with FVIII or HA protein (40 nM) were used to load AAPCs for 1 h prior to stimulation of the respective specific CD4+ T cell clones (A). For control experiments, AAPCs were incubated with 40 nM of FVIII or HA protein for 12 h and 6 h, respectively. Frequencies of activated CD4+ T cell clones were determined by ICS. The percentages of CD4+/IFN-?+ T cells are indicated in each FACS dot plots. AAPCs previously treated with different concentrations of inhibitors of MHC class II processing (B) dynasore (or its solvent) or (C) NH.sub.4Cl, were loaded with 40 nM of FVIII or HA protein for 12 h and 6 h, respectively before stimulation of the respective specific T cell clones. Results are expressed as percentages of IFN-?+ cells among CD4+ T lymphocytes.

[0067] FIG. 4: Priming of CD4 T cells by AAPCs or PBMCs.

[0068] AAPC.sup.DR1 pulsed with 10 ?g/ml of HA peptide were used to stimulate purified CD4+ T cells for 8-10 days. Alternatively, autologous PBMCs were cultured with 10 ?g/ml of HA peptide for the same duration. Frequency of HA-specific CD4+ effector T cells was evaluated by DR1-HA (or control DR1-CLIP) tetramer staining (A). ICS was performed after re-activation by AAPCs or B-EBV cell lines loaded with HA or control peptide for 6 h (B). Results show percentages of HA-tetramer+ or IFN-?+ cells among CD4+ T lymphocytes. A representative experiment is shown from five independent experiments with four donors.

[0069] FIG. 5: Restimulation of Ag-specific memory CD4 T cells by AAPCs or autologous PBMCs.

[0070] CD4+ T cells generated after primary culture of PBMCs with HA peptide for 7 days have been restimulated for 7 additional days with either AAPC.sup.DR1 or autologous PBMCs (aPBMCs) loaded or not with 10 ?g/ml of HA peptide. At day 0, 7 and 14, the percentages and the absolute numbers of HA-specific T cells were evaluated by tetramer staining and ICS. (A) A representative experiment of ICS performed after a 6 h re-activation by AAPCs or B-EBV cell lines loaded with HA or control peptide. Data of tetramer staining (B) and ICS (C) are from five independent experiments with four donors. *=p<0.05 (Student's paired t test), ns=not significant.

[0071] FIG. 6: Na?ve/memory phenotype of CD4 T cells.

[0072] PBMCs at day 0 (A), effector T cells collected after primary culture of PBMCs with HA peptide for 7 days (B) or after re-stimulation of primary effectors with AAPC.sup.DR1 loaded with HA peptide (day 14) (C) were stained with DR1-HA tetramer and with anti-CD4, CD45RA, CD45RO, CCR7, CD62L, CD122 and CD95 mAbs. Frequencies of na?ve and memory subsets are represented on FACS dot plots. A representative experiment is shown from five independent experiments with four donors. (D) Frequencies of CD122+ memory cells among CD4+/CD45RO+ T cells gated in DR1-HA tetramer negative or tetramer positive were analyzed before (day 0) and after first PBMC stimulation or re-stimulation with AAPC.sup.DR1 or autologous PBMCs. Data are from four independent experiments with five donors. *=p<0.005 (Student's paired t test)

EXAMPLE

[0073] Material & Methods

Healthy Subjects and of CD4+ T Cell Purification

[0074] Peripheral blood from HLA-DR1*01:01.sup.+ healthy donors of the French Blood Service (EFS Normandie, Caen, France) were collected in heparinized tubes after informed consent. PBMCs were isolated by density gradient centrifugation on lymphocyte separation medium (PAA Laboratories GmbH, Velizy-Villacoublay, France). CD4+ T cells were isolated from PBMCs by negative magnetic purification with CD4+ T cell isolation kit (Miltenyi Biotec, Paris, France) according to the manufacturer's instructions.

[0075] Construction of AAPCs

[0076] NIH/3T3-derived class II-AAPCs were constructed in the same way as NIH/3T3-derived class I-AAPCs we previously described [16],[17]. Briefly, cDNAs encoding HLA-DR?, HLA-DR?1*01:01, HLA-DR?1*15:01 and HLA-DR?5*01:01 chains were kindly provided by Dr. Klaus Dornmair (Institute of Clinical Neuroimmunology, Ludwig Maximilians University, Munich, Germany) in RSV expression vectors. The cDNAs were then cloned into gammaretrovirus-derived SFG vectors, between XhoI and BamHI sites. All the constructs were verified by DNA sequencing. Gammaretrovirus-derived SFG vectors encoding the human ICAM-1 (CD54), LFA-3 (CD58), and B7.1 (CD80) molecules were used for NIH/3T3-derived class I-AAPC construction. H29/293 GPG packaging cells were transfected with each vector by the calcium chloride precipitation method. NIH/3T3 cells were genetically modified by sequential infections with cell-free gammaretroviral supernatants corresponding respectively to B7-1, ICAM-1, LFA-3, HLA-DR? and HLA-DR? molecules, in the presence of 8 ?g/mL of polybrene (Sigma-Aldrich, Saint-Quentin Fallavier, France) for 16 hours. AAPCs, as NIH/3T3 cells, were then cultured in DMEM (Gibco Laboratories, Grand Island, N.Y.) with 10% of decomplemented AB serum (EFS Normandie).

[0077] Peptide and Protein Antigens

[0078] Three peptides were used: human coagulation FVIII .sub.2144IIARYIRLHPTHYSIRST.sub.2161 peptide (SEQ ID NO:1), HA .sub.306PKYVKQNTLKLAT.sub.318 peptide (SEQ ID NO:2) of H3N2 influenza virus and MBP .sub.84DENPVVHFFKNIVTPRTPP.sub.102 peptide (SEQ ID NO:3). These peptides bind HLA-DR51, HLA-DR1 and HLA-DR15 molecules respectively and were kindly provided by J. Leprince (Inserm U982, Rouen, France). The whole recombinant FVIII protein was a kind gift of Y. Repess? (Department of Biological Hematology, Caen University Hospital, France) and rHA protein (subtype H3N2, A/Aichi/2/1968) was purchased from Life Technologies (Saint-Aubin, France).

[0079] T Cell Clones and B-EBV Cell Lines

[0080] CD4+ T cells clones D9:E9 specific for FVIII.sub.2144-2161 peptide, Flu-2 specific for HA.sub.306-318 peptide and Ob1A12 specific for MBP.sub.84-102 peptide were kindly provided by M. Jacquemin (Center for Molecular and Vascular Biology, Louvain, Belgium), A. Godkin (Institute of Infection and Immunity, Cardiff, UK) and K. Wucherpfenning (Dana Faber Cancer Institute, Boston, Mass.), respectively [35-37]. The homozygous HLA-DR1 or HLA-DR15 B-EBV cell lines were kind gifts from and H. Vi? (Inserm U892, Nantes, France). Culture of T cell clones was performed in 96-well U bottom plates in RPMI supplemented with 1% of FBS (PAA Laboratories GmbH), 2 mM of glutamine, penicillin (50 IU/ml) and streptomycin (50 ?g/ml).

[0081] Cell Membrane Staining

[0082] Phenotypic expression of transduced molecules on AAPCs was determined by staining for 20-30 minutes at 4? C. in PBS/BSA buffer with the following Abs: FITC-conjugated anti-human LFA-3 and B7.1, PE-conjugated anti-human ICAM-1 (all three from Becton Dickinson, BD, Le Pont de Claix, France), unconjugated primary anti-HLA-DR complex Ab (Santa Cruz Biotechnology, Heidelberg, Germany) and revealed by FITC-conjugated anti-mouse IgG Ab (Jackson ImmunoResearch, Baltimore, Md.).

[0083] CD4+ T cells were stained with V500-conjugated anti-CD4 mAb. The na?ve/memory phenotype of T cells were studied by staining with PE-Cy7-conjugated anti-CD45RO, FITC-conjugated anti-CD62L, V450-conjugated anti-CD95, Alexa 647-conjugated anti-CCR7 (all from BD), APC (Allophycocyanin)/eFluor-780-conjugated anti-CD45RA (eBio science, Paris, France) and PerCP/Cy5.5-conjugated anti-CD122 (BioLegend, London, UK) mAbs. Frequency of HA-specific CD4 T cells was assessed by tetramer staining for 30 minutes at room temperature with PE-coupled with HLA-DRB1*01:01-HA (DR1-HA) or control HLA-DRB1*01:01-CLIP (DR1-CLIP) complexes (a kind gift of the NIH Tetramer Core Facility, Atlanta, Ga.). Cells were analyzed using FACSCantoII cytometer (BD) and Diva software (BD).

[0084] Stimulation of T Cell Clones and Intracellular Cytokine Staining

[0085] For evaluation of antigen presentation, CD4+ T cell clones (10.sup.5 per well) were co-cultured for 6 h with AAPC.sup.DR or B-EBV cell lines (10.sup.5 per well) loaded with different concentrations of FVIII, HA or control peptides. For protein presentation, plated AAPC.sup.DR or B-EBV cell lines were incubated with different concentrations of FVIII protein for 12 h or HA protein for 6 h. To analyze the presentation pathway in AAPCs, plated AAPC.sup.DR or B-EBV cell lines were treated with different concentrations of dynasore, its solvent control (DMSO) or NH.sub.4Cl (Sigma-Aldrich) before incubation with proteins. Then, CD4+ T cell clones (10.sup.5 per well) were co-cultured for 6 h with AAPC.sup.DR or B-EBV cell lines (10.sup.5 per well) whether previously treated or not with drugs, and incubated with the different proteins. Brefeldin A at 10 ?g/ml (Sigma-Aldrich) was added for the last 5 h of incubation and T cells were then fixed with paraformaldehyde (PFA 4%) prior to permeabilization in PBS/BSA/0.05% saponin buffer. CD4+ T cell clones were stained with PE-Cy7-conjugated anti-CD4 and APC-conjugated anti-IFN-? (Miltenyi Biotec).

[0086] Stimulation of MBP-Specific T Cell Clone and Proliferation Assay

[0087] For peptide presentation, MBP-specific T cell clone (10.sup.5 per well) was co-cultured 6 h with irradiated (35 Gy) AAPC.sup.DR15 or (70 Gy) B-EBV.sup.DR15 cell line (10.sup.4 per well) previously incubated 1 h with different concentrations of MBP or control peptides. For protein presentation, irradiated AAPC.sup.DR15 or B-EBV.sup.DR15 cell line (10.sup.4 per well) were incubated with different concentrations of MBP protein for 24 h. Then, MBP-specific T cell clone (10.sup.5 per well) was added for 48 h. Cultures were pulsed with 1 ?Ci of 3H-TdR (PerkinElmer, Villebon-sur-Yvette, France) for the last 16 h. Plates were harvested and measured in a scintillation counter (TopCount, Packard, Meriden, Conn.). Results are expressed as cpm?SD of triplicate cultures.

[0088] Stimulation of Purified CD4 T Cells by AAPCs or PBMCs

[0089] For primary stimulation experiments, irradiated AAPC.sup.DR1 loaded for 1 h with 10 ?g/ml of HA peptide were plated (1.5?10.sup.5 per well) 4 h before incubation with purified CD4+ T cells (10.sup.6 per well) for 8-10 days. Alternatively, PBMCs (2?10.sup.6 per well) were incubated with 10 ?g/ml of HA peptide for the same duration. For re-stimulation experiments, effector T cells (10.sup.6 per well) generated by primary culture of PBMCs with HA peptide were incubated with either irradiated adherent AAPC.sup.DR1 (1.5?10.sup.5 per well) or autologous PBMCs (2?10.sup.6 per well) whether or not loaded with 10 ?g/ml of HA peptide for 7 days. Cultures were performed in 24-well plates with AIM-V CTS medium (Life technologies) supplemented with 2 mM of glutamine and 5% of decomplemented AB serum. On day 3 and then, every other day, 20 IU/ml of IL-2 (Proleukin?, Chiron, Emeryville, Calif.) and 25 ng/ml of IL-7 (R&D systems, Lille, France) were added.

[0090] Statistics

[0091] All statistics were performed using GraphPad Prism 5 software (GraphPad Software Inc., La Jolla, Calif.) We used a paired, two-tailed Student's test, with p<0.05 considered significant.

[0092] Results

[0093] AAPC.sup.DR Stably Expressed Molecules Involved in Human CD4 T Cell Stimulation.

[0094] AAPC.sup.DR were constructed by transduction of murine fibroblasts NIH/3T3 with HLA-DR?, co-stimulatory B7.1 and adherence ICAM-1 and LFA-3 molecules. Transduction of NIH/3T3 cells was then completed with HLA-DRB*01:01 (HLA-DR1), HLA-DRB5*01:01 (HLA-DR51) or HLA-DRB1*15:01 (HLA-DR15) chains. AAPCs have been stained with anti-human HLA-DR, B7.1, ICAM-1 and LFA-3 Abs, fluorescence hatched histograms of the expression levels of transduced molecules in AAPCs after transduction or 3 months of culture have been analysed. Following transduction, AAPCs had high expression levels of HLA-DR, B7.1, ICAM-1 and LFA-3 molecules. Expression levels were stable for at least 3 months of continuous culture. B-EBV cell lines were also stained as a reference expression level in human APCs. B-EBV cell lines expressed higher levels of HLA-DR molecules but not of co-stimulatory or adherence molecules as compared with AAPC.sup.DR.

[0095] AAPC.sup.DR Efficiently Present Peptides and Process Proteins.

[0096] To assess the ability of AAPCs to present antigens, we had to our disposal three CD4+ T cell clones that recognize the epitopes factor VIII (FVIII).sub.2144-2161, HA.sub.306-318, or myelin basic protein (MBP).sub.84-102 epitope. AAPC.sup.DR51 or AAPC.sup.DR1 loaded with FVIII or HA peptides, respectively, were able to activate specific T cell clones as assessed by intracellular cytokine staining (ICS) of IFN-?. T cells specific for FVIII or HA peptides did not recognize the unmatched AAPC.sup.DR15 loaded cells. AAPC.sup.DR15 loaded with MBP peptide stimulated proliferation of a specific T cell clone as measured by incorporation of tritiated thymidine (H3-TdR) (FIG. 1C). Peptide titration experiments with each of the 3 Ags showed that AAPC.sup.DR were more effective to present peptides than their respective HLA matched B-EBV cell lines (FIGS. 1A, B and C).

[0097] AAPC.sup.DR51 or AAAPC.sup.DR1 loaded with whole FVIII or HA protein, respectively, were also able to stimulate T cell clones (FIGS. 2A and B). Optimal time of incubation was 6 and 12 h for HA and FVIII proteins, respectively. We did not observe any significant difference in T cell clone activation with protein loaded B-EBV cell lines. To evaluate protein processing and presentation by AAPCs, FVIII or HA protein was maintained in medium or added to AAPCs for 16 h under the same conditions (FIG. 3A). Then, other plated AAPCs were incubated for 1 h with the medium containing proteins or the supernatant of AAPC incubated with proteins, prior to addition to T cell clones. Under both conditions, activation of T cell clones was minimal compared to the control with peptide loaded AAPC excluding significant extra-cellular degradation in the medium or by AAPCs.

[0098] Mechanisms of antigen presentation were further investigated using inhibitors of MHC class II processing pathway. AAPCs were first treated with dynasore, an inhibitor of endocytosis, and loaded with FVIII or HA protein. Under these conditions with these 2 Ags, AAPCs were unable to present epitopes to T cells (FIG. 3B). Similarly, treatment with NH.sub.4Cl which prevents acidification of endosomal compartment also inhibited antigen presentation by AAPCs for both proteins (FIG. 3C). These data show that AAPCs were able to present peptide and to effectively process whole proteins generating relevant peptides for human T cells.

[0099] AAPC.sup.DR are Able to Stimulate CD4 T Cells from Healthy Donors.

[0100] The viral HA.sub.306-318 epitope for which specific T cells can be detected in healthy donors was used to assess the ability of AAPCs to stimulate CD4 T cell response in primary culture [18]. Purified CD4+ T cells (>92% of purity) from donors were stimulated for 8-10 days with AAPC.sup.DR1 loaded with HA peptide. Frequency of specific T cells among generated T cells was evaluated by tetramer staining or ICS after re-activation by AAPCs or B-EBV cell lines. AAPC.sup.DR1 were able to generate HA-specific T cells with about 2% of CD4+/HA-tetramer+ T cells (FIG. 4A). This result was confirmed by IFN-? production with 1.6% of CD4+/IFN-?+ T cells after reactivation by B-EBV.sup.DR1 loaded with HA as compared to 0.1% with control peptide (FIG. 4B). Surprisingly, a high frequency of CD4+/IFN-?+ T cells (about 30% of CD4+ T cells) was observed after reactivation by AAPC.sup.DR1 pulsed with HA or control peptides. These HA-nonspecific effector T cells, likely xenoreactive T cells, were however restricted by HLA-DR1 as they were not activated by with HA peptide pulsed AAPC.sup.DR15. When PMBCs from the same donors were directly stimulated with HA peptide, a higher frequency of specific CD4+ T cells was obtained with about 7% of HA-tetramer+ T cells and 5% of CD4+/IFN-?+ T cells. Therefore, AAPCs were able to activate specific CD4+ T cells although some irrelevant molecules were also presented to human T cells.

[0101] AAPCs Restimulate Ag-Specific Memory CD4+ T Cells Better than PBMCs.

[0102] We then compared the capacity of AAPC.sup.DR1 or autologous PBMCs to re-stimulate memory CD4+ T cells generated after primary stimulation of total PBMCs with HA peptide. Restimulation of primary effector T cells with irradiated AAPC.sup.DR1 or autologous PBMCs pulsed with HA peptide increased the frequency of HA-specific T cells by 2 to 3 fold (FIG. 5). The percentage of CD4+/IFN-?+ and HA-tetramer+ T cells were similar for the two types of presenting cells with about 30 to 40% of HA-specific T cells. However, the absolute number of effector cells was significantly increased after re-stimulation with AAPCs with a 30 fold increase as compared to 18 with PBMC (p=0.035). Restimulation of primary CD4+ T cells with unloaded AAPC.sup.DR1 did not modify the frequency of effector T cells ruling out any major bystander feeder effect. Importantly, restimulation of effectors with AAPC.sup.DR1 did not generate non-relevant T cells as observed previously in primary cultures. In ICS experiments, reactivation by AAPC.sup.DR1 pulsed with HA peptide showed a high frequency of CD4+/IFN-?+ that were absent after reactivation with the control peptide (FIG. 5A).

[0103] The na?ve/memory phenotype of CD4+ T cells was carefully studied. In freshly isolated PBMCs, we observed a low frequency of HA-specific T cells (less than 1% of CD4+/HA-tetramer+ T cells) which were mainly CD45RO+ memory T cells (FIG. 6A). Among these memory specific CD4+ T cells, effector memory (EM) cells (CCR7?/CD62L?) were the main populations. Other memory cells were central memory (CM) cells (CCR7+/CD62L+) and a transitional memory (TM) population (CCR7?/CD62L+). All memory CD4+ T cells expressed CD95 but not CD122. This pattern was similar to CD4+ T cells not activated by HA peptide. In na?ve CD4+/HA-tetramer-/CD45RA+ T cells, we observed a major contingent of CCR7+/CD62L+ cells that did not express either CD95 or CD122. After stimulation of PBMCs with HA, all specific CD4+ T cells expressed CD45RO, had a predominant TM phenotype and expressed CD122 de novo (FIG. 6B). This was observed in all donors tested (FIG. 6D). Among CD4+/HA-tetramer negative T cells, we predominantly observed CM and TM cells as well as a population of EM cells that were still CD122 negative. The restimulation of effector T cells by AAPC.sup.DR1 or autologous PBMCs did not substantially modify the phenotype. Nevertheless, CD122 expression was strongly reduced on specific memory CD4+ T cells (FIG. 6C). Finally, CD122 expression was significantly increased after specific stimulation of PBMCs but only transiently.

[0104] Finally, the inventors also showed that specific memory regulatory T cells (Tregs) purified from circulating CD4+/CD25+ T cells (Thymic Tregs) and primed by Ag-loaded APCs in presence of rapamycin and IL-2 could be amplified by AAPCs in the same conditions. The same method is also usable to expand induced Treg from purified na?ve CD4+/CD25? T cells.

[0105] Discussion

[0106] The multiple properties of CD4+ T cells open new opportunities not only for immunotherapy in chronic viral infections and cancer but also for severe autoimmune diseases and transplantation [4, 11, 19, 20]. Efficient antigen-driven expansion is critical for the development of CD4+ T cell based adoptive transfer. Therefore, we were prompted to develop an AAPC system engineered to express molecules involved in the immunological synapse including HLA class II, co-stimulatory and adhesion molecules in cells not able to present Ag. Using three antigen models, we showed that AAPCs loaded with peptides strongly activate specific T cell clones even at higher levels than professional presenting cells such as B-EBV cells. Since the CD28 pathway is involved in T cell activation, stronger expression of B7.1 molecules on AAPC.sup.DR than B-EBV cells may explain the superior stimulating capacity of AAPC.sup.DR [21].

[0107] AAPC.sup.DR are also able to process and present immunogenic CD4+ T cell epitopes derived from full-length proteins as shown with HA and FVIII antigens. We furthermore demonstrated that this antigenic presentation of exogenous proteins resulted from endocytosis and trafficking in endosomes as in professional APC and not from extrinsic antigen degradation. In our previously reported studies, we have shown that AAPC derived from the same NIH/3T3 cell backbone and bearing human MHC class I molecules present epitopes derived from viral or tumor antigens and generate CTL with potent effector functions [16, 17, 22]. Overall, our data illustrate similarities in MHC class I and II antigen processing pathways between human and murine cells making NIH/3T3-derived AAPCs an appropriate platform to stimulate or monitor both CD8 and CD4 responses against multiple known or unknown epitopes in different HLA backgrounds.

[0108] AAPC.sup.DR1 pulsed with an epitope of the viral HA Ag triggered expansion of specific CD4+ T cells from HLA-matched healthy donors as illustrated by tetramer staining. AAPC.sup.DR also expanded effector CD4+ T cells that recognize non-relevant epitopes in an HLA-DR-restricted context. It is likely that these non-relevant epitopes derive from endogenous murine proteins and/or medium contained Ags. Typically, the MHC class II molecules present peptides from the extracellular environment but also endogenous antigens derived from intracellular organites via autophagy mechanism or recycling of cell surface molecules [1]. Targeting expression of relevant epitopes into endosomes where antigens are degraded and loaded on MHC class II molecules can be an attractive approach to optimize specific antigen presentation and reduce irrelevant murine presentation by AAPC.sup.DR [23]. Indeed, several studies have used protein sorting signals to reach endosomal compartments such as the melanosomal protein GP75 or lysosome associated membrane protein-1 (LAMP-1). APC genetically modified with vectors encoding antigenic peptides or whole proteins fused with endosomal targeting motif have been shown to enhance CD4+ T cell responses in vitro or in vivo [24, 25]. Such approaches will be investigated in a near future.

[0109] Primary stimulation of purified CD4+ T cells with HA peptide using AAPC.sup.DR generated lower specific effector T cells in terms of percentage and absolute number than direct stimulation of PBMC, probably due to competition with xenoantigens. Nevertheless, we were able to demonstrate that memory CD4+ T cells restimulated with AAPC.sup.DR underwent a more robust expansion than with autologous PBMC resulting in a 2 to 3 fold increased expansion than with PBMCs. Interestingly, restimulation with AAPC.sup.DR did not trigger expansion of non-relevant T cells as observed in primary culture. This makes AAPC.sup.DR a suitable and reliable tool to amplify memory CD4+ T cells in an Ag-dependent manner.

[0110] Tetramer-based analyses of in vitro human CD4+ T cell responses are still rarely reported [18, 26]. In our study, the phenotype of CD4+ T cells before and after one or two round(s) of specific stimulation was carefully analyzed by eight-color flow cytometry using a reliable tetramer and a panel of Abs against cell surface differentiation markers. Prior to expansion, tetramer-specific CD4+ T cells represented less than 1% of CD4+ T cells and had an EM phenotype (CD45RO+/CCR7?/CD62L?) probably corresponding to antigen experienced T cells as shown for EBV-specific T cells in EBV-carrier subjects [26]. In addition, in our experiments, these cells expressed the Fas receptor (CD95) but not the interleukin-2 receptor beta chain (CD122). The recently described T cell subset termed stem cell memory T cells, with a capacity for self-renewal, and distinguished from na?ve T cell by high expression of CD95 and CD122 was undetectable among CD4+ T cells and represents less than 1% of the CD4 negative counterparts (data not shown) [27]. Interestingly, after in vitro primary culture of PBMCs with HA peptide, the great majority of HA-specific CD4+ T cells displayed a CCR7?/CD62L+ phenotype, typical of TM T cells. TM CD4+ T cells have been detected in healthy donors and HIV patients and have functional and transcriptional features which are intermediary between those of CM and EM T cells [28-30].

[0111] Interestingly, we reported for the first time to our knowledge that the CD122 receptor is expressed de novo and transiently only on antigen-specific CD4+ T cells. Thus, CD122 represents a cell surface marker for antigen-experienced T cells and may constitute a promising candidate for the identification and sorting of antigen-specific T cells as the T cell activation markers CD137 or CD154 [31, 32].

[0112] Only very few studies have investigated antigen-specific stimulation of human CD4+ T cells with AAPC systems. Hirano et al. have used the human erythroleukemia cell line K562 genetically modified to express HLA-DR, CD80 and CD83 molecules [33]. Although a valid comparison of both stimulation systems would require the use of the same Ags and the same functional assays, our AAPC model clearly showed several advantages. Our AAPC-based protocol enable the short term expansion of specific CD4+ T cells and the presentation of naturally processed epitope from exogenous protein, in a side by side comparison with other conventional APCs such as autologous PBMCs and B-EBV cell lines.

[0113] In addition to enable fundamental studies on the processing of the MHC class II pathway and the characterization of novel CD4+ T cell epitopes, AAPC could also be modified to express different costimulatory and/or inhibitory molecules potentially involved in the different Th and/or regulatory CD4+ T cells responses.

[0114] Clinical trials for adoptive T cell therapy require large numbers of T cells, at least 10.sup.9 to 10.sup.11 cells [10, 34]. Based on a conservative estimation of 8-fold expansion obtained after primary stimulation of PBMCs followed by restimulation with AAPC.sup.DR, the rapid generation of 10.sup.9 effector CD4+ T cells would require about 1.3?10.sup.8 peripheral blood CD4+ T cells as the basic materiel, thus approximately 300 ml of blood.

[0115] In conclusion, we were able to establish a protocol allowing reproducible and effective amplification of Ag-specific CD4+ T cells using a novel AAPC system. Our expansion protocol may be applied to clinically relevant Ags and pave the way for future immunotherapy strategies. In addition, our AAPC system is also a useful model to dissect any type of CD4 responses and may represent a useful tool to identify novel epitopes naturally processed in any HLA context.

REFERENCES

[0116] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. [0117] 1. Neefjes J, Jongsma M L M, Paul P, Bakke O. Towards a systems understanding of MHC class I and MHC class II antigen presentation. Nat. Rev. Immunol. 2011; 11:823-836. [0118] 2. Yamane H, Paul W E. Early signaling events that underlie fate decisions of naive CD4(+) T cells toward distinct T-helper cell subsets. Immunol. Rev. 2013; 252:12-23.DOI: 10.1111/imr.12032. [0119] 3. Josefowicz S Z, Lu L-F, Rudensky A Y. Regulatory T cells: mechanisms of differentiation and function. Annu. Rev. Immunol. 2012; 30:531-564.DOI: 10.1146/annurev.immunol.25.022106.141623. [0120] 4. Kamphorst A O, Ahmed R. CD4 T-cell immunotherapy for chronic viral infections and cancer. Immunotherapy. 2013; 5:975-987.DOI: 10.2217/imt.13.91. [0121] 5. Wilkinson T M, Li C K F, Chui C S C, Huang A K Y, Perkins M, Liebner J C, Lambkin-Williams R, et al. Preexisting influenza-specific CD4+ T cells correlate with disease protection against influenza challenge in humans. Nat. Med. 2012; 18:274-280.DOI: 10.1038/nm.2612. [0122] 6. Smyk-Pearson S, Tester I A, Klarquist J, Palmer B E, Pawlotsky J-M, Golden-Mason L, Rosen H R. Spontaneous recovery in acute human hepatitis C virus infection: functional T-cell thresholds and relative importance of CD4 help. J. Virol. 2008; 82:1827-1837. [0123] 7. Einsele H, Roosnek E, Rufer N, Sinzger C, Riegler S, L?ffler J, Grigoleit U, et al. Infusion of cytomegalovirus (CMV)-specific T cells for the treatment of CMV infection not responding to antiviral chemotherapy. Blood. 2002; 99:3916-3922. [0124] 8. Perruccio K, Tosti A, Burchielli E, Topini F, Ruggeri L, Carotti A, Capanni M, et al. Transferring functional immune responses to pathogens after haploidentical hematopoietic transplantation. Blood. 2005; 106:4397-4406.DOI: 10.1182/blood-2005-05-1775. [0125] 9. Muranski P, Restifo N P. Adoptive immunotherapy of cancer using CD4(+) T cells. Curr. Opin. Immunol. 2009; 21:200-208.DOI: 10.1016/j.coi.2009.02.004. [0126] 10. Tran E, Turcotte S, Gros A, Robbins P F, Lu Y-C, Dudley M E, Wunderlich J R, et al. Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer. Science. 2014; 344:641-645.DOI: 10.1126/science.1251102. [0127] 11. Singer B D, King L S, D'Alessio F R. Regulatory T cells as immunotherapy. Front. Immunol. 2014; 5:46.DOI: 10.3389/fimmu.2014.00046. [0128] 12. Tarbell K V, Petit L, Zuo X, Toy P, Luo X, Mqadmi A, Yang H, et al. Dendritic cell-expanded, islet-specific CD4+CD25+CD62L+ regulatory T cells restore normoglycemia in diabetic NOD mice. J. Exp. Med. 2007; 204:191-201.DOI: 10.1084/jem.20061631. [0129] 13. Stephens L A, Malpass K H, Anderton S M. Curing CNS autoimmune disease with myelin-reactive Foxp3+ Treg. Eur. J. Immunol. 2009; 39:1108-1117. [0130] 14. Sagoo P, Ali N, Garg G, Nestle F O, Lechler R I, Lombardi G. Human regulatory T cells with alloantigen specificity are more potent inhibitors of alloimmune skin graft damage than polyclonal regulatory T cells. Sci. Transl. Med. 2011; 3:83ra42.DOI: 10.1126/scitranslmed.3002076. [0131] 15. Kim J V, Latouche J-B, Rivi?re I, Sadelain M. The ABCs of artificial antigen presentation. Nat. Biotechnol. 2004; 22:403-410.DOI: 10.1038/nbt955. [0132] 16. Papanicolaou G A, Latouche J-B, Tan C, Dupont J, Stiles J, Pamer E G, Sadelain M. Rapid expansion of cytomegalovirus-specific cytotoxic T lymphocytes by artificial antigen-presenting cells expressing a single HLA allele. Blood. 2003; 102:2498-2505.DOI: 10.1182/blood-2003-02-0345. [0133] 17. Fauquembergue E, Toutirais O, Tougeron D, Drouet A, Le Gallo M, Desille M, Cabillic F, et al. HLA-A*0201-restricted CEA-derived peptide CAP1 is not a suitable target for T-cell-based immunotherapy. J. Immunother. Hagerstown Md. 1997. 2010; 33:402-413.DOI: 10.1097/CJI.0b013e3181d366da. [0134] 18. Scriba T J, Purbhoo M, Day C L, Robinson N, Fidler S, Fox J, Weber J N, et al. Ultrasensitive detection and phenotyping of CD4+ T cells with optimized HLA class II tetramer staining. J. Immunol. Baltim. Md. 1950. 2005; 175:6334-6343. [0135] 19. Zanetti M. Tapping CD4 T cells for cancer immunotherapy: the choice of personalized genomics. J. Immunol. Baltim. Md. 1950. 2015; 194:2049-2056.DOI: 10.4049/jimmunol.1402669. [0136] 20. Issa F, Chandrasekharan D, Wood K J. Regulatory T cells as modulators of chronic allograft dysfunction. Curr. Opin. Immunol. 2011; 23:648-654.DOI: 10.1016/j.coi.2011.06.005. [0137] 21. Gimmi C D, Freeman G J, Gribben J G, Gray G, Nadler L M. Human T-cell clonal anergy is induced by antigen presentation in the absence of B7 costimulation. Proc. Natl. Acad. Sci. U.S.A 1993; 90:6586-6590. [0138] 22. Dupont J, Latouche J-B, Ma C, Sadelain M. Artificial antigen-presenting cells transduced with telomerase efficiently expand epitope-specific, human leukocyte antigen-restricted cytotoxic T cells. Cancer Res. 2005; 65:5417-5427.DOI: 10.1158/0008-5472.CAN-04-2991. [0139] 23. Boudreau J E, Bonehill A, Thielemans K, Wan Y. Engineering dendritic cells to enhance cancer immunotherapy. Mol. Ther. J. Am. Soc. Gene Ther. 2011; 19:841-853. [0140] 24. Wang S, Bartido S, Yang G, Qin J, Moroi Y, Panageas K S, Lewis J J, et al. A role for a melanosome transport signal in accessing the MHC class II presentation pathway and in eliciting CD4+ T cell responses. J. Immunol. Baltim. Md. 1950. 1999; 163:5820-5826. [0141] 25. Bonini C, Lee S P, Riddell S R, Greenberg P D. Targeting antigen in mature dendritic cells for simultaneous stimulation of CD4+ and CD8+ T cells. J. Immunol. Baltim. Md. 1950. 2001; 166:5250-5257. [0142] 26. Long H M, Chagoury O L, Leese A M, Ryan G B, James E, Morton L T, Abbott R J M, et al. MHC II tetramers visualize human CD4+ T cell responses to Epstein-Barr virus infection and demonstrate atypical kinetics of the nuclear antigen EBNA1 response. J. Exp. Med. 2013; 210:933-949.DOI: 10.1084/jem.20121437. [0143] 27. Gattinoni L, Klebanoff C A, Restifo N P. Paths to stemness: building the ultimate antitumour T cell. Nat. Rev. Cancer. 2012; 12:671-684.DOI: 10.1038/nrc3322. [0144] 28. Riou C, Yassine-Diab B, Van grevenynghe J, Somogyi R, Greller L D, Gagnon D, Gimmig S, et al. Convergence of TCR and cytokine signaling leads to FOXO3a phosphorylation and drives the survival of CD4+ central memory T cells. J. Exp. Med. 2007; 204:79-91.DOI: 10.1084/jem.20061681. [0145] 29. Chomont N, El-Far M, Ancuta P, Trautmann L, Procopio F A, Yassine-Diab B, Boucher G, et al. HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation. Nat. Med. 2009; 15:893-900.DOI: 10.1038/nm.1972. [0146] 30. Flynn J K, Paukovics G, Cashin K, Borm K, Ellett A, Roche M, Jakobsen M R, et al. Quantifying susceptibility of CD4+ stem memory T-cells to infection by laboratory adapted and clinical HIV-1 strains. Viruses. 2014; 6:709-726.DOI: 10.3390/v6020709. [0147] 31. Khanna N, Stuehler C, Conrad B, Lurati S, Krappmann S, Einsele H, Berges C, et al. Generation of a multipathogen-specific T-cell product for adoptive immunotherapy based on activation-dependent expression of CD154. Blood. 2011; 118:1121-1131. [0148] 32. Ye Q, Song D-G, Poussin M, Yamamoto T, Best A, Li C, Coukos G, et al. CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 2014; 20:44-55. [0149] 33. Butler M O, Ans?n S, Tanaka M, Imataki O, Berezovskaya A, Mooney M M, Metzler G, et al. A panel of human cell-based artificial APC enables the expansion of long-lived antigen-specific CD4+ T cells restricted by prevalent HLA-DR alleles. Int. Immunol. 2010; 22:863-873.DOI: 10.1093/intimm/dxq440. [0150] 34. Hunder N N, Wallen H, Cao J, Hendricks D W, Reilly J Z, Rodmyre R, Jungbluth A, et al. Treatment of metastatic melanoma with autologous CD4+ T cells against NY-ESO-1. N. Engl. J. Med. 2008; 358:2698-2703.DOI: 10.1056/NEJMoa0800251. [0151] 35. Jacquemin M, Vantomme V, Buhot C, Lavend'homme R, Bumy W, Demotte N, Chaux P, et al. CD4+ T-cell clones specific for wild-type factor VIII: a molecular mechanism responsible for a higher incidence of inhibitor formation in mild/moderate hemophilia A. Blood. 2003; 101:1351-1358.DOI: 10.1182/blood-2002-05-1369. [0152] 36. Cole D K, Gallagher K, Lemercier B, Holland C J, Junaid S, Hindley J P, Wynn K K, et al. Modification of the carboxy-terminal flanking region of a universal influenza epitope alters CD4+ T-cell repertoire selection. Nat. Commun. 2012; 3:665. [0153] 37. Wucherpfennig K W, Sette A, Southwood S, Oseroff C, Matsui M, Strominger J L, Hafler D A. Structural requirements for binding of an immunodominant myelin basic protein peptide to DR2 isotypes and for its recognition by human T cell clones. J. Exp. Med. 1994; 179:279-290.