WO2016180852A1 - Methods for preparing antigen-specific t cells from an umbilical cord blood sample - Google Patents

Methods for preparing antigen-specific t cells from an umbilical cord blood sample Download PDF

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WO2016180852A1
WO2016180852A1 PCT/EP2016/060500 EP2016060500W WO2016180852A1 WO 2016180852 A1 WO2016180852 A1 WO 2016180852A1 EP 2016060500 W EP2016060500 W EP 2016060500W WO 2016180852 A1 WO2016180852 A1 WO 2016180852A1
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cells
il
method
specific
ag
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French (fr)
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Roberto Mallone
Sophie CAILLAT-ZUCMAN
Klaudia KURANDA
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Université Paris Descartes
Centre National De La Recherche Scientifique (Cnrs)
Université Paris Diderot - Paris 7
Universite De Paris-Sud
Assistance Publique-Hôpitaux De Paris (Aphp)
Association Robert Debré
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Application filed by INSERM (Institut National de la Santé et de la Recherche Médicale), Université Paris Descartes, Centre National De La Recherche Scientifique (Cnrs), Université Paris Diderot - Paris 7, Universite De Paris-Sud, Assistance Publique-Hôpitaux De Paris (Aphp), Association Robert Debré filed Critical INSERM (Institut National de la Santé et de la Recherche Médicale)
Publication of WO2016180852A1 publication Critical patent/WO2016180852A1/en

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    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues ; Not used, see subgroups
    • C12N5/0602Vertebrate cells
    • C12N5/0634Cells from the blood or the immune system
    • C12N5/0636T lymphocytes
    • C12N5/0638Cytotoxic T lymphocytes [CTL], lymphokine activated killer cells [LAK]
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    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues ; Not used, see subgroups
    • C12N5/0602Vertebrate cells
    • C12N5/0634Cells from the blood or the immune system
    • C12N5/0639Dendritic cells, e.g. Langherhans cells in the epidermis
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    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20Cytokines; Chemokines
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    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20Cytokines; Chemokines
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    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20Cytokines; Chemokines
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    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20Cytokines; Chemokines
    • C12N2501/26Flt-3 ligand (CD135L, flk-2 ligand)
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    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50Cell markers; Cell surface determinants
    • C12N2501/52CD40, CD40-ligand (CD154)

Abstract

The present invention relates methods for preparing antigen- specific T cells from an umbilical cord blood sample. In particular, the present invention relates to a method for preparing Ag-specific T cells from a CB sample isolated from a subject comprising the steps of i) culturing said CB sample in an appropriate culture medium, which comprises an amount of at least one agent capable of stimulating dendritic cell differentiation and an amount of at least one Ag and ii) isolating said Ag-specific T cells.

Description

METHODS FOR PREPARING ANTIGEN-SPECIFIC T CELLS FROM AN

UMBILICAL CORD BLOOD SAMPLE

FIELD OF THE INVENTION:

The present invention relates methods for preparing antigen- specific T cells from an umbilical cord blood sample.

BACKGROUND OF THE INVENTION:

The development of techniques for propagating T cell populations in vitro has been crucial to many of the recent advances in the understanding of T cell recognition of antigens (Ags) and T cell activation. The development of culture methods for the generation of human Ag-specific T cell clones has been useful in defining Ags expressed by pathogens and tumors that are recognized by T cells to establish methods of immunotherapy to treat a variety of human diseases. For instance, Ag-specific T cells can be expanded in vitro for use in adoptive cellular immunotherapy, in which infusions of such T cells have been shown to have antitumor reactivity in a tumor-bearing host. Adoptive immunotherapy has also been used to treat viral infections in immunocompromised individuals (Fujita Y et al. Bone Marrow Transplant 2008). Donor leukocyte infusions (DLI) in the allogeneic hematopoietic transplant setting can provide a clinically relevant boost of immunity to reduce opportunistic infections and to increase graft-versus-leukemia activity. Despite significant advances in applicability, DLI has not been available for the growing number of recipients of hematopoietic stem cell transplantation (HSCT) from unrelated cord blood (CB) donors. While HSCT from a HLA- matched sibling remains the first choice, this option is available for only 30% of patients. Finding a suitably matched unrelated donor is increasingly difficult in today's multiethnic society. When no fully matched donor is found, three allogeneic HSCT sources are available: related haplo-identical donors, unrelated mismatched donors and unrelated CB units. Key advantages of CB HSCT are its ready availability; its tolerance for some degree of HLA mismatch, which greatly helps in finding suitable CB units; and a reduced risk of graft- versus-host disease (GvHD) and relapse. On the other hand, both engraftment and immune recovery are delayed, due to lower numbers of stem and T cells and to the larger proportion of naive T cells than what is obtained from adult sources; and only a single or few CB units per donor are usually available, without the possibility of referring to the original CB donor for further procedures. These factors contribute to post-transplant infections and related mortality (1). Nonetheless, partially matched CB HSCT gives similar long-term leukemia-free survival compared to fully matched unrelated bone marrow HSCT in both children (2) and adults (3).

One major drawback of all HSCT is the period of immune deficiency which precedes immune reconstitution, which lasts longer in CB HSCT recipients. This period leaves transplanted patients vulnerable to infections from viruses such as cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus (AdV, mainly AdV5). Such infections mostly occur within the first 6 months after HSCT. While antiviral agents (ganciclovir, foscarnet) and anti- CD20 treatment can often control CMV reactivation and EBV-associated lymphoproliferation respectively, such agents are toxic and not always effective, and no effective treatment (with the limited exception of cidofovir) is available in the case of AdV (4). AdV infection is particularly daunting in children, occurring in 10-30% of cases and in up to 80% in children under 5 years of age (4). Overall, intercurrent infections score as the first contributor to CB HSCT-related mortality, due to the longer time before immune reconstitution and, probably, to the immunologically "immature" state of CB T cells (5).

Adoptive T-cell therapy is frequently used to control these infections and sometimes used to control leukemia relapses in the context of HSCT from adult donors. In several centers, DLIs are performed to mount T-cell responses against threatening infections. Typically, the HSC donor is admitted to the hospital to undergo leukapheresis. Peripheral blood mononuclear cells (PBMCs) thus obtained are stimulated overnight with a mixture of antigenic peptides (Miltenyi PepTivator) derived from the virus of interest (mostly AdV or CMV). Virus- specific T cells are subsequently magnetically sorted based on their IFN-γ secretion using an IFN-γ capture assay (Miltenyi) (6), and this fraction is infused intravenously to the HSCT recipient. The cell needs for this type of procedure is of ~109 PBMCs to obtain l-50xl03 Ag-specific T cells/kg to infuse into patients (6). Both CD4+ and CD8+ T cells are needed for optimal viral clearance (6). This approach is however made difficult by the delay needed to re-contact the donor when this is not a patient's relative. Another approach used for EBV reactivation is the generation of T cells by stimulation with donor's EBV-transformed B cells during a 15-day culture (7). Regulatory rules may however make these procedures difficult to implement on a large scale. Biobanks of ready-to-use Ag- specific T cells obtained from third-party allogeneic donors harboring common HLA haplotypes are also being proposed, but this approach is currently limited to EBV. Moreover, other studies showed that these T cells, despite failure to induce GvHD, did not persist post- transfer and multiple infusions were required for effective treatment, suggesting that they may eventually be rejected by the recipients (7). Thus, all current procedures share the same limitations, namely the need to use PBMCs from the original HSC donor to limit the occurrence of GvHD; and the requirement for a large number of starting PBMCs. These two features make these procedures unfeasible in the setting of CB HSCT, for which the CB donor is not available and the CB unit(s) used for HSCT are limited in amount. One further challenge is that the CB T-cell repertoire is largely Ag-inexperienced, hence requiring to expand Ag- specific T cells from naive precursors. These drawbacks currently limit both wider applicability of CB HSCT and the success rate in patients on whom the procedure is performed.

Hence, it is highly desirable to develop techniques allowing to generate viral- specific T cells in sufficient quantities for adoptive cell therapies, starting from the limited cell numbers available in CB unit(s), without jeopardizing the success of concomitant HSCT using the same CB unit(s). In the few works addressing this issue (8, 9), CB T cells were successfully expanded in 8-14 days starting from a negligible (3-5%) fraction of single CB units. However, expansion was not obtained with viral Ags, but with polyclonal anti- CD3/CD28 bead stimulation, which yields a higher risk of GvHD. On the other hand, investigators that successfully obtained viral Ag- specific T cells from CB did so by using large numbers of starting cells (>40xl06, i.e. >20% of a typical CB unit) stimulated with crude Ag sources such as CMV lysates, B-EBV lines and transduced Ag-presenting cells (APCs) (10-12), quenching enthusiasm towards clinical application.

We have recently developed an accelerated co-cultured dendritic cell (acDC) technology which may meet these requirements (13). Using appropriate cytokine cocktails, this technology allows to differentiate and mature DCs directly in situ, using unfractionated PBMCs cultured in vitro without preliminary purification of monocytes or other DC precursors. Such differentiation and maturation can be achieved within a 48 h culture period. When Ag stimuli such as peptides, proteins or whole cells are further added at the start of culture, T-cell precursors (both CD4+ and CD8+, either memory or naive) recognizing these Ags are stimulated and can be efficiently expanded over the next few (9-11) days and sorted for further use. The three key advantages of this technology are that: 1) PBMC needs to obtain such Ag-specific T cells are significantly reduced (typically only 1-2 million PBMCs per Ag specificity are needed); 2) Ag-specific T cells are efficiently amplified, as the procedure lines up the three critical steps of Ag processing, presentation and T-cell triggering both spatially, within a small culture well, and temporally (typically 9-11 days to obtain significant expansion of the desired Ag-specific T-cell fractions); 3) the use of professional APCs such as DCs allows to use a variety of Ag stimuli, including long peptide fragments, without prior knowledge of the precise epitopes recognized and without selecting donors based on specific HLA haplotypes needed for efficient epitope presentation. WO2010119033 and WO2014173858 describe methods for generating and testing antigen- specific T cells from a blood sample by using acDCs. However said documents do not describe or anticipate that acDCs could be applied to cord blood sample for preparing antigen- specific T cells.

SUMMARY OF THE INVENTION:

The present invention relates methods for preparing antigen- specific T cells from an umbilical cord blood sample. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

The inventors now demonstrated that the accelerated co-cultured dendritic cell (acDC) technology is suitable for preparing antigen (Ag)-specific T cells from an umbilical cord blood (CB) sample.

Thus, the invention relates to a method for preparing Ag-specific T cells from a CB sample isolated from a subject comprising the steps of i) culturing said CB sample in an appropriate culture medium, which comprises an amount of at least one agent capable of stimulating dendritic cell differentiation and an amount of at least one Ag and ii) isolating said Ag-specific T cells.

As used herein the term "umbilical cord blood" or "CB" has its general meaning in the art and refers to blood that remains in the placenta and in the attached umbilical cord after child birth. CB contains stem cells including hematopoietic cells, and more specifically CD34+ cells. The CB sample is typically obtained from fresh CB supplemented with an anticoagulant, reconstituted cryopreserved cord blood or a fresh or reconstituted cryopreserved mononuclear cell fraction thereof. In some embodiments, the process for obtaining a CB sample according to the invention involves red blood cell depletion of the CB, further subjected to density gradient separation to isolate the mononuclear fraction. For instance, the CB-derived mononuclear cells can be isolated by a known method, e.g., a modified Ficoll- Hypaque method, a 3% gelatin method, a Ficoll-Hypaque method (Kim et al., Optimal umbilical cord blood processing: Basic study for the establishment of cord blood bank, Korean Journal of Hematopoietic Stem Cell Transplantation. 2000.5:61-68) and other procedures known to the expert in the art. The CB mononuclear fraction may then be subjected to CD marker selection (e.g; CD34+) by electronic or magnetic sorting. For instance, the mononuclear cells may be analyzed by a flow cytometer (e.g. FACSAria III, BD, U.S.A.) to determine the presence of stem cells and immune cells in the mononuclear cell fraction. Mononuclear cells derived from CB according to the present invention are present as immature cells and retain remarkable differentiation and proliferation capabilities. In some embodiments, the CB sample may be the fraction remaining after isolation of CD34+ cells for hematopoietic stem cell transplantation. The remaining CD34-negative fraction may then be subjected to the described acDC procedures to obtain Ag-specific T cells. Any culture medium suitable for growth, survival and differentiation of mononuclear cells may be used. Typically, it consists of a base medium containing nutrients (a source of carbon, amino acids), a pH buffer and salts, which can be supplemented with serum of human or other origin and/or growth factors and/or antibiotics to which cytokines and Ags are added. Typically, the base medium can be RPMI 1640, DMEM, EVIDM, X-VIVO or AIM-V medium, all of which are commercially available standard media.

In some embodiments, the agent capable of stimulating dendritic cell differentiation is a cytokine. As used herein the term "cytokine" has its general meaning in the art. Typically, examples of cytokines include lymphokines, interleukins, and chemokines.

As used herein the term "interleukin" has its general meaning in the art and refers to any interleukin (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL- 26, and IL-27) polypeptide. In some embodiments, the cytokine is selected from the group consisting of Interleukin 1 (IL-1), Interleukin 2 (IL-2), Interleukin 3 (IL-3), Interleukin 4 (IL- 4), Interleukin 5 (IL-5), Interleukin 6 (IL-6), Interleukin 7 (IL-7), Interleukin 8 (IL-8), Interleukin 9 (IL-9), Interleukin 10 (IL-10), Interleukin 11 (IL-11), Interleukin 12 (IL-12), Interleukin 13 (IL-13), Interleukin 15 (IL-15), and Interleukin 17 (IL-17) polypeptides.

In some embodiments, the culture medium comprises an amount of Granulocyte/Macrophage Colony-Stimulating Factor (GM-CSF) and an amount of interleukin (IL)-4. Typically, GM-CSF is used in an amount comprised between 1 and 10,000 U/ml, preferably between 10 and 5,000 U/ml, even more preferably at about 1,000 U/ml. GM-CSF can be obtained from a variety of sources. It may be purified or recombinant GM-CSF. GM- CSF is commercially available from different companies, for example R&D Systems or PeproTech. Typically, IL-4 is used in an amount comprised between 0 and 10,000 U/ml, preferably between 10 and 1,000 U/ml, even more preferably at about 500 U/ml. IL-4 can be obtained from a variety of sources. It may be purified or recombinant IL-4. IL-4 is commercially available from different companies, for example R&D Systems or PeproTech.

In some embodiments, the culture medium comprises an amount of FMS-like tyrosine kinase 3 (Flt-3) ligand. Flt-3 ligand may be used alone or in combination with GM-CSF and/or IL-4. Typically, Flt-3 ligand is used in an amount comprised between 1 and 1,000 ng/ml, preferably between 10 and 100 ng/ml. Flt-3 ligand can be obtained from a variety of sources. It may be purified or recombinant Flt-3 ligand. Flt-3 ligand is commercially available from different companies, for example R&D Systems or PeproTech.

In some embodiments, the culture medium comprises an amount of IL-Ιβ. As used herein, the term "IL-Ιβ" has its general meaning in the art and refers to interleukin-ΐβ. IL-Ιβ may be used alone, subsequently to or in combination with GM-CSF and/or IL-4 and/or Flt3 ligand. Typically, IL-Ιβ is used in an amount comprised between 0.1 and 1,000 ng/ml, preferably between 1 and 100 ng/ml, even more preferably at about 10 ng/ml. IL-Ιβ can be obtained from a variety of sources. It may be purified or recombinant IL-Ιβ. IL-Ιβ is commercially available from different companies, for example R&D Systems or PeproTech.

In some embodiments, said medium comprises pro-inflammatory stimuli and/or agents which mimic a viral or bacterial aggression. These molecules may be used alone, subsequently to or in combination with GM-CSF and/or IL-4 and/or Flt3 ligand and/or IL-Ιβ. They can be used as single agents or combinations thereof. Examples of pro-inflammatory stimuli suitable for the method of the invention are, but are not limited to, tumor necrosis factor alpha (TNF-a), prostaglandin E2 (PGE2), anti-CD40 monoclonal antibodies (mAbs), CD40 ligand (CD40L) recombinant chimeric proteins, interferon- alpha (IFN-a), interferon- gamma (IFN-γ), IL-7. Such agents can be used alone or in different combinations with other pro-inflammatory stimuli or viral/bacterial mimetic agents. Examples of agents which mimic a viral or bacterial aggression suitable for the method of the invention are, but are not limited to, lipopolysaccharides (LPS), CpG oligodeoxynucleotides, polyinosinic:polycytidylic acid (poly I:C), Pam3CysSerLys4 (Pam3CSK4), imiquimod. Such agents can be used alone or in different combinations with other pro-inflammatory stimuli or viral/bacterial mimetic agents. Said agent(s) are agents known to stimulate immune responses, and the skilled person will be able to select the appropriate concentrations of each agent for obtaining DCs while limiting non-specific T-cell activation. Also, the skilled person will easily construe that other agents which are known to stimulate DC differentiation can also be used according to the method of the invention.

In some embodiments, the agent capable of stimulating dendritic cell differentiation is a ligand suitable for the activation of a pathogen recognition receptor.

As used herein the term "pathogen recognition receptor" or "PRR" has its general meaning in the art and refers to a class of receptors expressed by cells of the innate immune system (including DCs, macrophages, mast cells and neutrophils) to identify pathogen- associated molecular patterns (PAMPs), which are associated with microbial pathogens or cellular stress, as well as damage- associated molecular patterns (DAMPs), which are associated with cell components released during cell damage. PPRs include membrane-bound PRRs (e.g. Receptor kinases, Toll-like receptors (TLR), C-type lectin Receptors) and cytoplasmic PRRs (e.g. NOD-like receptors (NLR), or RIG-Tlike receptors).

In some embodiments, the ligand that is suitable for the activation of a pathogen recognition receptor is a TLR agonist.

As used herein the term "Toll like receptor (TLR)" has its general meaning in the art and describes a member of the Toll-like receptor family of proteins or a fragment thereof that senses a microbial product and/or initiates an innate or an adaptive immune response. Tolllike receptors include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR 8, TLR9, TLR10, TR11 and TLR12. The term "agonist" as used herein in referring to a TLR activating molecule, means a molecule that activates a TLR signaling pathway. As discussed above, a TLR signaling pathway is an intracellular signal transduction pathway employed by a particular TLR that can be activated by the TLR agonist. Common intracellular pathways are employed by TLRs and include, for example, NF- κ B, Jun N-terminal kinase and mitogen- activated protein kinase. The TLR agonism for a particular compound may be assessed in any suitable manner. For example, assays for detecting TLR agonism of test compounds are described in U.S. Provisional Patent Application Ser. No. 60/432,650, filed Dec. 11, 2002; and recombinant cell lines suitable for use in such assays are described, for example, in U.S. Provisional Patent Application Ser. No. 60/432,651, filed Dec. 11, 2002. In one embodiment, the TLR agonist is selected from the group consisting of TLR1,

TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR 10, TLR11, TLR 12, or TLR 13 agonists. TLR agonists are well known in the art (see e.g. Baxevanis CN, Voutsas IF, Tsitsilonis OE. Toll-like receptor agonists: current status and future perspective on their utility as adjuvants in improving anticancer vaccination strategies. Immunotherapy, 2013 May; 5(5):497-511. doi: 10.2217/imt.13.24; Shaherin Basith, Balachandran Manavalan, Gwang Lee, Sang Geon Kim, Sangdun Choi Toll-like receptor modulators: a patent review (2006 - 2010) Expert Opinion on Therapeutic Patents Jun 2011, Vol. 21, No. 6, Pages 927- 944; 20. Heather L. Davis Chapter 26: TLR9 Agonists for Immune Enhancement of Vaccines, New Generation Vaccines, Fourth Edition; Jory R Baldridge, Patrick McGowan, Jay T Evans, Christopher Cluff, Sally Mossman, David Johnson, David Persing Taking a Toll on human disease: Toll-like receptor 4 agonists as vaccine adjuvants and monotherapeutic agents Expert Opinion on Biological Therapy Jul 2004, Vol. 4, No. 7, Pages 1129-1138.).

In one embodiment, the TLR agonist is a TLR1 agonist. Examples of TLR1 agonists include tri-acylated lipopeptides (LPs); phenol- soluble modulin; Mycobacterium tuberculosis LP; S-(2,3- bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys(4)-OH, trihydrochloride (Pam3Cys) LP which mimics the acetylated amino terminus of a bacterial lipoprotein and OspA LP from Borrelia burgdorferi. In one embodiment, the TLR agonist is a TLR2 agonist. For example, the TLR2 agonist consists of a flagellin modification protein FImB of Caulobacter crescentus; Bacterial Type III secretion system protein; invasin protein of Salmonella; Type 4 fimbrial biogenesis protein (PilX) of Pseudomonas; Salmonella SciJ protein; putative integral membrane protein of Streptomyces; membrane protein of Pseudomonas; adhesin of Bordetella pertusis; peptidase B of Vibrio cholerae; virulence sensor protein of Bordetella; putative integral membrane protein of Neisseria meningitidis; fusion of flagellar biosynthesis proteins FIiR and FHiB of Clostridium; outer membrane protein (porin) of Acinetobacter; flagellar biosynthesis protein FHiF of Helicobacter; ompA related protein of Xanthomonas; omp2a porin of Brucella spp.; putative porin/fimbrial assembly protein (LHrE) of Salmonella; wbdKK of Salmonella; Glycosyltransferase involved in LPS biosynthesis; Salmonella putative permease. In one embodiment, the TLR2 agonist is selected form the group consisting of lipoprotein/LPs (isolate from a variety of pathogens); peptidoglycan (isolated form Gram-positive bacteria); lipoteichoic acid (isolated from Gram- positive bacteria); lipoarabinomannan (isolated from mycobacteria); a phenol-soluble modulin (isolated from Staphylococcus epidermidis); glycoinositolphospholipids (isolated form Trypanosoma Cruzi); glycolipids (isolated from Treponema maltophilum); porins (isolated from Neisseria); zymosan (isolated from fungi) and atypical LPS (isolated form Leptospira interrogans and Porphyromonas gingivalis). The TLR2 agonist can also include at least one member disclosed in PCT/US 2006/002906/WO 2006/083706; PCT/US 2006/003285/WO 2006/083792; PCTAJS 2006/041865; PCT/US 2006/042051). The TLR2 agonist can include at least a portion of a bacterial lipoprotein (BLP), such as Pam2Cys (S-[2,3- bis(palmitoyloxy) propyl] cysteine), Pam3Cys ([Palmitoyl]- Cys((RS)-2,3- di(palmitoyloxy)-propyl cysteine) or Pseudomonas aeruginosa Oprl lipoprotein (Oprl). A bacterial lipoprotein that activates a TLR2 signaling pathway (a TLR2 agonist) is a bacterial protein that includes a palmitoleic acid (Omueti, K.O., et al, J. Biol. Chem. 280: 36616-36625 (2005)).

In one embodiment, the TLR agonist is a TLR3 agonist. For example, TLR3 agonists include naturally-occurring double-stranded RNA (dsRNA); synthetic dsRNA; and synthetic dsRNA analogs; and the like (Alexopoulou et al, 2001). An exemplary, non- limiting example of a synthetic dsRNA analog is Poly(LC).

In one embodiment, the TLR agonist of the invention is a TLR4 agonist. Various TLR4 agonists are known in the art, including Monophosphoryl lipid A (MPLA), in the field also abbreviated to MPL, referring to naturally occurring components of bacterial lipopolysaccharide (LPS); refined detoxified endotoxin. For example, MPL is a derivative of lipid A from Salmonella minnesota R595 lipopolysaccharide (LPS or endotoxin). While LPS is a complex heterogeneous molecule, its lipid A portion is relatively similar across a wide variety of pathogenic strains of bacteria. MPL, used extensively as a vaccine adjuvant, has been shown to activate TLR4 (Martin M. et al., 2003. Infect Immun. 71(5):2498-507; Ogawa T. et al., 2002. Int Immunol. 14(11): 1325-32). TLR4 agonists also include natural and synthetic derivatives of MPLA, such as 3-de-O-acylated monophosphoryl lipid A (3D- MPL), and MPLA adjuvants available from Corixa Corporation (Seattle, WA; see US Patents 4,436,727; 4,436,728; 4,987,237; 4,877,611; 4,866,034 and 4,912,094 for structures and methods of isolation and synthesis). A structure of MPLA is disclosed in US 4,987,237. Nontoxic diphosphoryl lipid A (DPLA) may also be used, for example OM-174, a lipid A analogue of bacterial origin containing a triacyl motif linked to a diglucosamine diphosphate backbone. Another class of useful compounds are synthetic lipid A analogue pseudo- dipeptides derived from amino acids linked to three fatty acid chains (see for example EP 1242365), such as OM-197-MP-AC, a water soluble synthetic acylated pseudo-dipeptide (C55H107N4O12P). Non-toxic TLR4 agonists include also those disclosed in EP1091928, PCT/FR05/00575 or PCT/IB2006/050748. PCT/US2006/002906/WO 2006/083706; PCT/US 2006/003285/WO 2006/083792; PCT/US 2006/041865; PCT/US 2006/042051. TLR4 agonists also include synthetic compounds which signal through TLR4 other than those based on the lipid A core structure, for example an aminoalkyl glucosaminide 4-phosphate (see Evans JT et al. Expert Rev Vaccines. 2003 Apr;2(2):219-29; or Persing et al. Trends Microbiol. 2002; 10(10 Suppl):S32-7. Review). Other examples include those described in Orr MT, Duthie MS, Windish HP, Lucas EA, Guderian J A, Hudson TE, Shaverdian N, O'Donnell J, Desbien AL, Reed SG, Coler RN. MyD88 and TRIF synergistic interaction is required for THl-cell polarization with a synthetic TLR4 agonist adjuvant. Eur J Immunol. 2013 May 29. doi: 10.1002/eji.201243124.; Lambert SL, Yang CF, Liu Z, Sweetwood R, Zhao J, Cheng L, Jin H, Woo J. Molecular and cellular response profiles induced by the TLR4 agonist-based adjuvant Glucopyranosyl Lipid A. PLoS One. 2012;7(12):e51618. doi: 10.1371/journal.pone.0051618. Epub 2012 Dec 28.

In one embodiment, the TLR agonist is a TLR5 agonist. Typically, the TLR5 agonist according to the invention is a flagellin polypeptide. As used herein, the term "flagellin" is intended to mean the flagellin contained in a variety of Gram-positive or Gram-negative bacterial species. Sources of flagellins include but are not limited to Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella enterica serovar Typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces. These examples are illustrative rather than limiting. The amino acid sequences and nucleotide sequences of flagellins are publically available in the NCBI Genbank, see for example Accession Nos. AAL20871, NP_310689, BAB58984, AA085383, AAA27090, NP_461698, AAK58560, YP_001217666, YP_002151351, YP_001250079, AAA99807, CAL35450, AAN74969, and BAC44986. The flagellin sequences from these and other species are intended to be encompassed by the term flagellin as used herein. Therefore, the sequence differences between species are included within the meaning of the term. The term "flagellin polypeptide" is intended to comprise a flagellin or a fragment thereof that retains the ability to bind and activate TLR5. Examples of flagellin polypeptides include but are not limited to those described in U.S. Pat. Nos. 6,585,980; 6, 130,082; 5,888,810; 5,618,533; and 4,886,748; U.S. Patent Publication No. US 2003/0044429 Al; and in the International Patent Application Publications n° WO 2008097016 and WO 2009156405, which are incorporated by reference.

In one embodiment, the TLR agonist is a TLR7 agonist. For example, TLR7 agonists include, but are not limited to: imidazoquinoline-like molecules, imiquimod, resiquimod, gardiquimod, S-27609; and guanosine analogues such as loxoribine (7-allyl-7,8-dihydro-8- oxo-guanosine), 7-Thia-8-oxoguanosine and 7-deazaguanosine, UC- 1V150, ANA975 (Anadys Pharmaceuticals), SM-360320 (Sumimoto), 3M-01 and 3M-03 (3M Pharmaceuticals) (see for example Gorden et al., J Immunol, 2005; Schon et al., Oncogene, 2008; Wu et al., PNAS 2007). TLR7 agonists include imidazoquinoline compounds; guanosine analogs; pyrimidinone compounds such as bropirimine and bropirimine analogs; and the like. Imidazoquinoline compounds that function as TLR7 ligands include, but are not limited to, imiquimod (also known as Aldara, R-837, S-26308), and R-848 (also known as resiquimod, S-28463; having the chemical structure: 4-amino-2-ethoxymethyl- a , a . - dimethyl- 1 H- imidazol[4,5-c]quinoline-l-ethanol). Suitable imidazoquinoline agents include imidazoquinoline amines, imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine amines, and 1,2 bridged imidazoquinoline amines.

In one embodiment, the TLR agonist is a TLR8 agonist. TLR8-selective agonists include those in U.S. Patent Publication 2004/0171086. Such TLR8 selective agonist compounds include, but are not limited to, the compounds shown in U.S. Patent Publication No. 2004/0171086 that include N-{4-[4-amino- 2-(2-methoxyethyl)-lH-imidazo[4,5- c]quinolin-l-yl]butyl}quinolin-3-carboxamide, N-{4-[4- amino-2-(2-methoxyethyl)-lH- imidazo[4,5-c]quinolin-l-yl]butyl}quinoxoline-2- carboxamide, and N-[4-(4-amino-2 -propyl- lH-imidazo[4,5-c]quinolin-l- yl)butyl]morpholine-4-carboxamide. Other suitable TLR8- selective agonists include, but are not limited to, 2- propylthiazolo[4,5-c]quinolin-4-amine (U.S. Patent 6,110,929); Nl-[2-(4-amino-2-butyl-lH- imidazo[4,5-c] [ 1 ,5]naphthyridin- 1 - yl)ethyl]~2-amino-4-methylpentanamide (U.S. Patent 6, 194,425); Nl-[4-(4-amino-lH- imidazo[4,5-c]quinolin-l-yl)butyl]-2-phenoxy-benzamide (U.S. Patent 6,451,810); Nl-[2-(4- amino-2-butyl-lH-imidazo[4,5-c]quinolin-l-yl)ethyl]-l- propanesulfonamide (U.S. Patent 6,331,539); N-{2-[2-(4-amino-2-ethyl-lH-imidazo[4,5- c]quinolin-l-yl)ethyoxy] ethyl }- N'~phenylurea (U.S. Patent Publication 2004/0171086); l-{4-[3,5- dichlorophenyl)thio]butyl}-2-ethyl-lH-imidazo[4,5-c]quinolin-4~ amine (U.S. Patent Publication 2004/0171086); N- {2-[4-amino-2-(ethoxymethyl)-lH-imidazo[4,5-c]quinolin- 1 - yl]ethyl }-N'-(3-cyanophenyl)urea (WO 00/76518 and U.S. Patent Publication No. 2004/0171086); and 4-amino- a , a -dimethyl-2-methoxyethyl- lH-imidazo[4,5-c]quinoline-l - ethanol (U.S. Patent 5,389,640). Included for use as TLR8- selective agonists are the compounds in U.S. Patent Publication No. 2004/0171086. Also suitable for use is the compound 2-propylthiazolo-4,5-c]quinolin-4-amine.

In a particular embodiment, the TLR agonist is a TLR9 agonist. Examples of TLR9 agonists include nucleic acids comprising the sequence 5'-CG-3' (a "CpG nucleic acid"), where C maybe unmethylated. The terms "polynucleotide," and "nucleic acid", as used interchangeably herein in the context of TLR9 agonist molecules, refer to a polynucleotide of any length, and encompasses, inter alia, single- and double- stranded oligonucleotides (including deoxyribonucleotides, ribonucleotides, or both), modified oligonucleotides, and oligonucleosides, alone or as part of a larger nucleic acid construct, or as part of a conjugate with a non-nucleic acid molecule such as a polypeptide. Thus a TLR9 agonist may be, for example, single- stranded DNA (ssDNA), double- stranded DNA (dsDNA), single-stranded RNA (ssRNA) or double- stranded RNA (dsRNA). TLR9 agonists also encompass crude, detoxified bacterial (e.g., mycobacterial) RNA or DNA, as well as plasmids enriched for a TLR9 agonist. In some embodiments, a "TLR9 agonist-enriched plasmid" refers to a linear or circular plasmid that comprises or is engineered to comprise a greater number of CpG motifs than normally found in mammalian DNA. Examples of non-limiting TLR9 agonist-enriched plasmids are described in Roman et al. (1997). In general, a TLR9 agonist used in a subject composition comprises at least one unmethylated CpG motif. In some embodiments, a TLR9 agonist comprises a central palindromic core sequence comprising at least one CpG sequence, where the central palindromic core sequence contains a phosphodiester backbone, and where the central palindromic core sequence is flanked on one or both sides by phosphorothioate backbone-containing polyguanosine sequences. In other embodiments, a TLR9 agonist comprises one or more TCG sequences at or near the 5' end of the nucleic acid; and at least two additional CG dinucleotides. In some of these embodiments, the at least two additional CG dinucleotides are spaced three nucleotides, two nucleotides, or one nucleotide apart. In some of these embodiments, the at least two additional CG dinucleotides are contiguous with one another. In some of these embodiments, the TLR9 agonist comprises (TCG)n, where n = 1 to 3, at the 5' end of the nucleic acid. In other embodiments, the TLR9 agonist comprises (TCG)n, where n = 1 to 3, and where the (TCG)n sequence is flanked by one nucleotide, two nucleotides, three nucleotides, four nucleotides, or five nucleotides, on the 5' end of the (TCG)n sequence. A TLR9 agonist of the present invention includes, but is not limited to, any of those described in U.S. Patent Nos. 6,194,388; 6,207,646; 6,239,116; 6,339,068; and 6,406,705, 6,426,334 and 6,476,000, and published US Patent Applications US 2002/0086295, US 2003/0212028, and US 2004/0248837. In some embodiments, the ligand that is suitable for the activation of a pathogen recognition receptor is a NOD-like receptor ligand. The NOD-like receptor ligand can be without limitation selected from the group consisting of NODI, NOD2, IPAF, Nalplb, and Cryopirin/Nalp3 ligand. The NOD-like receptor ligand is preferably meso-diaminopimelic acid, muramyl dipeptide or flagellin. Alternatively, the NOD-like receptor ligand is NODI, NOD2, IPAF, Nalpl b or Cryopirin/Nalp3 ligand.

In some embodiments, the culture medium is supplemented with at least one homeostatic cytokine. Typically, the cytokine is selected from IL-2, IL-7 and IL-15, or combinations thereof.

According to the invention, step i) is performed for an amount of time sufficient for enriching the CB sample in dendritic cells and Ag-specific T cells. Thus, the step is carried out for an amount of time t(i) comprised between t(i)min and t(i)max. Typically, the minimal incubation for step i), t(i)min, can be about 12 hours, preferably about 24 hours, even more preferably about 48 hours. Typically, the maximum incubation for step i), t(i)max can be about 21 days, even more preferably about 14 day. In some embodiments, step i) is carried out for an amount of time t(i) of about 10 days.

The term "antigen" ("Ag") as used herein refers to protein, peptide, nucleic acid (e.g. DNA plasmid) or tissue or cell preparations capable of eliciting a T-cell response. In some embodiments, said Ag is a protein which can be obtained by recombinant DNA technology or by purification from different tissue or cell sources. Such proteins are not limited to natural ones, but also include modified proteins or chimeric constructs, obtained for example by changing selected amino acid sequences or by fusing portions of different proteins. In some embodiments, said Ag is a synthetic peptide, obtained by Fmoc biochemical procedures, large-scale multipin peptide synthesis, recombinant DNA technology or other suitable procedures. In some embodiments, said Ag is a protein or peptide coded by a DNA or other suitable nucleic acid sequence which has been introduced in cells by transfection, lentiviral or retroviral transduction, mini-gene transfer or other suitable procedures. In some embodiments, the Ag is a crude tissue or cell preparation (e.g., live cells or apoptotic cells/bodies) or a partially purified tissue or cell preparation obtained by different biochemical procedures (e.g., fixation, lysis, subcellular fractionation, density gradient separation) known to the expert in the art. In some embodiments, said Ag is a protein which can be obtained by recombinant DNA technology or by purification from different tissue or cell sources. Typically, said protein has a length higher than 10 amino acids, preferably higher than 15 amino acids, even more preferably higher than 20 amino acids with no theoretical upper limit. Such proteins are not limited to natural ones, but also include modified proteins or chimeric constructs, obtained for example by changing selected amino acid sequences or by fusing portions of different proteins. In some embodiments, said Ag is a synthetic peptide. Typically, said synthetic peptide is 3-40 amino acid-long, preferably 5-30 amino acid-long, even more preferably 8-20 amino acid-long. Synthetic peptides can be obtained by Fmoc biochemical procedures, large- scale multipin peptide synthesis, recombinant DNA technology or other suitable procedures. Such peptides are not limited to natural ones, but also include modified peptides, post- translationally modified peptides or chimeric peptides, obtained for example by changing or modifying selected amino acid sequences or by fusing portions of different proteins. In some embodiments, said Ag is a protein or peptide coded by a DNA or other suitable nucleic acid sequence which has been introduced in cells by transfection, lentiviral or retroviral transduction, mini-gene transfer or other suitable procedures. The recipient cells may be either third party cells (e.g., cell lines obtained from the same CB sample or from unrelated donors) or the same cells present in the CB sample used for stimulating T-cell responses. In some embodiments, the Ag is a tissue or cell preparation (e.g., live cells or apoptotic cells/bodies) or a crude or partially purified tissue or cell preparation obtained by different biochemical procedures (e.g., fixation, lysis, subcellular fractionation, density gradient separation) known to the expert in the art. The skilled person in the art will be able to select the appropriate Ag, depending on the desired T-cell stimulation.

In some embodiments, the Ag is a viral Ag. Examples of viral Ags include but are not limited to influenza viral Ags (e.g. hemagglutinin (HA) protein, matrix 2 (M2) protein, neuraminidase), respiratory syncitial virus (RSV) Ags (e.g. fusion protein, attachment glycoprotein), polio, papillomaviral (e.g. human papilloma virus (HPV), such as an E6 protein, E7 protein, LI protein and L2 protein), Herpes simplex, rabies virus and flavivirus viral Ags (e.g. Dengue viral Ags, West Nile viral Ags), hepatitis viral Ags including Ags from HBV and HCV, human immunodeficiency virus (HIV) Ags (e.g. gag, pol or nef), herpesvirus (such as cytomegalovirus and Epstein-Barr virus) Ags (e.g. pp65, IE1, EBNA-1, BZLF-1) and adenovirus Ags.

In some embodiments, the antigen is a bacterial Ag. Examples of bacterial Ags include but are not limited to those from Streptococcus pneumonia, Haemophilus influenza, Staphylococcus aureus, Clostridium difficile and enteric gram-negative pathogens including Escherichia, Salmonella, Shigella, Yersinia, Klebsiella, Pseudomonas, Enterobacter, Serratia, Proteus, B. anthracis, C tetani, B. pertussis, S. pyogenes, S. aureus, N. meningitidis and Haemophilus influenzae type b.

In some embodiments, the Ag is a fungal or protozoal Ag. Examples include but are not limited to those from Candida spp., Aspergillus spp., Crytococcus neoformans, Coccidiodes spp., Histoplasma capsulatum, Pneumocystis carinii, Paracoccidiodes brasiliensis, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae.

In some embodiments, the Ag is a tumor-associated Ag (TAA). Examples of TAAs include, without limitation, melanoma-associated Ags (Melan-A/MART-1, MAGE-1, MAGE-3, TRP-2, melanosomal membrane glycoprotein gplOO, gp75 and MUC-1 (mucin-1) associated with melanoma); CEA (carcinoembryonic Ag) which can be associated, e.g., with ovarian, melanoma or colon cancers; folate receptor alpha expressed by ovarian carcinoma; free human chorionic gonadotropin beta (hCGP) subunit expressed by many different tumors, including but not limited to ovarian tumors, testicular tumors and myeloma; HER-2/neu associated with breast cancer; encephalomyelitis antigen HuD associated with small-cell lung cancer; tyrosine hydroxylase associated with neuroblastoma; prostate-specific antigen (PSA) associated with prostate cancer; CA125 associated with ovarian cancer; and the idiotypic determinants of a B-cell lymphoma that can generate tumor- specific immunity (attributed to idiotype-specific humoral immune response). Moreover, Ags of human T cell leukemia virus type 1 have been shown to induce specific cytotoxic T cell responses and anti-tumor immunity against the virus-induced human adult T-cell leukemia (ATL). Other leukemia Ags can equally be used.

In some embodiments, the method of the invention further comprises a step consisting of detecting stimulated T cells. Methods for the detection of stimulated T cells are known to the skilled person. In some embodiments, said method may consist in an enzyme-linked immunospot (ELISpot) assay. Non-adherent cells from pre-culture wells are transferred to a plate which has been coated with the desired anti-cytokine capture antibodies (Abs; e.g., anti- IFN-γ, -IL-10, -IL-2, -IL-4). Revelation is carried out with biotinylated secondary Abs and standard colorimetric or fluorimetric detection methods such as streptavidin-alkaline phosphatase and NBT-BCIP and the spots counted. In some embodiments, said method may consist in a supernatant cytokine assay. Cytokines released in the culture supernatant are measured by different techniques, such as enzyme-linked immunosorbent assays (ELISA), BD cytometric bead array, Biorad, Millipore or Meso Scale Discovery cytokine multiplex assays and others. All said methods are suitable for detecting the T cells of interest.

In some embodiments, the method may use HLA Class I or Class II multimers. With this procedure, Ag-reactive T cells recognizing specific peptide epitopes are detected, using either commercially available reagents (e.g., Prolmmune MHC Class I Pentamers, Class II Ultimers; or Immudex MHC Dextramers) or in-house generated ones, e.g., from the NIH Tetramer Facility at Emory University, USA; from Dr. S. Buus, University of Copenhagen, Denmark [Leisner et al., PLoSOne 3:el678, 2008], from Dr. G.T. Nepom, Benaroya Research Institute, Seattle, USA [Novak et al., J.Clin.Invest. 104:R63, 1999]. In some embodiments, the method is based on the detection of the upregulation of activation markers (e.g., CD69, CD25, CD137). With this procedure, Ag-specific T cell responses are detected by their differential expression of activation markers exposed on the membrane following Ag- recognition. In some embodiments, the method may consist in a cytokine capture assay. This system developed by Miltenyi Biotech is a valid alternative to the ELISpot to visualize Ag- specific T cells according to their cytokine response. In some embodiments, the method may consist of a CD154 assay. This procedure has been described in detail [Chattopadhyay et al., Nat.Med. 11:1113, 2005; Frentsch et al., Nat.Med. 11: 1118, 2005]. It is limited to detection of Ag-specific CD4+ T cells. In some embodiments, the method may consist in a CD107 assay. This procedure [Betts et al., J.Immunol.Methods 281:65, 2003] allows the visualization of Ag-specific CD8+ T cells with cytotoxic potential. In some embodiments, the method may consist in a CFSE dilution assay. This procedure detects Ag-specific T cells (CD4+ and CD8+) according to their proliferation following Ag recognition [Mannering et al., J.Immunol.Methods 283: 173, 2003]. Other methods suitable for detecting cell proliferation (e.g. BrdU incorporation, Ki67 expression) may also be used. Besides being suitable for detecting Ag-specific T cells, said methods allows the direct sorting and/or cloning of the T cells of interest (see below).

In some embodiments, it is desirable to select the Ag-specific T cells generated by said procedures in order to obtain preparations of higher purity. The person skilled in the art is familiar with the methods described above that are suitable to isolate said Ag-specific T cells in a viable state based on different immunological properties. For example, selection of IFN- γ- or IL-10-producing T cells may be obtained by Miltenyi cytokine capture assays. As another example, selection of cytotoxic T cells may be obtained based on upregulation of CD107 [Betts et al., J.Immunol.Methods 281:65, 2003] or other suitable markers of activation (including, but not limited to, CD69, CD25, CD127, CD154 and combinations thereof) or proliferation (including, but not limited to, CFSE, BrdU, Ki67). As yet another example, said T cells can be isolated by means of MHC Class I or Class II multimers [Mallone et al., Diabetes 53:971, 2004; Martinuzzi et al., Blood 106:2798, 2005; Skowera et al., J.Clin.Invest. 118:3390, 2008; Afonso et al., J.Immunol.Methods 359:28, 2010; Scotto et al., Diabetes 61:2546, 2012; Ladell et al., Immunity 38:425, 2013].

Once isolated, the Ag-specific T cells prepared by the method of the present invention are expanded. The person skilled in the art is familiar with methods for expanding said Ag- specific T cells. Examples of such methods can be found in Reijonen et al., Diabetes 51: 1375, 2002; Martinuzzi et al., Blood 106:2798, 2005; Mannering et al., J.Immunol.Methods 298:83, 2005; Yee et al., J.Immunol. 162:2227, 1999; Mandruzzato et al., J.Immunol. 169:4017, 2002; Oelke et al., Nat.Med. 9:619, 2003; Skowera et al., J.Clin.Invest. 118:3390, 2009.

The antigen- specific T cells prepared by the method of the present invention are particularly suitable for adoptive immunotherapy in subjects in need thereof.

For example, the Ag-specific T cells of the present invention are suitable for the treatment of cancer. As used herein, the term "cancer" has its general meaning in the art and includes, but is not limited to, solid tumors and blood-borne tumors. The term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels. The term "cancer" further encompasses both primary and metastatic cancers. Examples of cancers that may be treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adeno squamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

In some embodiments, the Ag-specific T cells prepared by the method of the present invention are suitable for treating subjects afflicted with, or at risk of developing, an infectious disease, including but not limited to viral, retroviral, bacterial, and protozoal infections, etc. Subjects that can be treated include immunodeficient patients afflicted with a viral infection, including but not limited to CMV, EBV, adenovirus, BK polyomavirus infections in transplant patients, etc. Typically, the subjects at risk of developing an infectious disease include patients undergoing hematopoietic stem cell transplantation using peripheral blood or CB precursors. As used herein, the term "patient undergoing hematopoietic stem cell transplantation (HSCT)" refers to a human being who has to be transplanted with HSC graft. Typically, said patient is affected with a disorder which can be cured by HSCT. In some embodiments, the patient undergoing HSCT is affected with a disorder selected from the group consisting of leukemia, lymphoma, myeloproliferative disorders, myelodysplasia syndrome (MDS), bone marrow (BM) failure syndromes, congenital immunodeficiencies, enzyme deficiencies and hemoglobinopathies. In some embodiments, the HSCT is an allogeneic HSCT. As used herein, the term "allogeneic" refers to HSC deriving from, originating in, or being members of the same species, where the members are genetically related or genetically unrelated but genetically similar. An "allogeneic transplant" refers to transfer of cells or organs from a donor to a recipient, where the recipient is the same species as the donor. Allogeneic transplantation involves infusion of donor stem cells, typically using a donor that matches the recipient's MHC. However, matched unrelated donor (MUD) transplants are also associated with a stronger graft versus host reaction, and thus result in higher mortality rates. In another embodiment, the HSCT is an autologous HSCT. As used herein, the term "autologous" refers to deriving from or originating in the same subject or patient. An "autologous transplant" refers to collection and retransplant of a subject's own cells or organs. Autologous transplantation involves infusion of a recipient's own cells following myeloablative treatment. Autologous cell transplants minimize the risk of graft versus host disease (GVHD) and result in reduced complications. Thus, the Ag-specific T cells prepared by the method of the present invention are particularly suitable for preventing bacterial, viral, protozoal and/or fungal infection following CB HSCT. Non- limiting examples of viral infections include Herpes simplex virus (HSV) infections, CMV infections, Varicella- zoster virus (VZV) infections, Human herpes virus 6 (HHV6) infections, EBV infections, respiratory virus infections (such as respiratory syncytial virus (RSV), parainfluenza virus, rhinovirus, and influenza virus) and adenovirus infections. Non-limiting examples of bacterial infections include Gram-negative bacteria infections such as Escherichia (e.g. Escherichia coli), Salmonella, Shigella, and other Enterobacteriaceae, Pseudomonas (e.g. Pseudomonas aeruginosa), Moraxella, Helicobacter, and Legionella infections. Non-limiting examples of protozoal infections include Giardia infections (e.g. Giardia lamblia), Entamoeba infections (e.g. Entamoeba histolytica) and Toxoplasma (e.g. Toxoplasma gondii). Non-limiting examples of fungal infections include Aspergillus infection (e.g. Aspergillus fumigatus), Candida infection (e.g. Candida albicans and non-albicans Candida) and other emerging fungal infections including Trichosporon, Alternaria, Fusarium, and Mucorales infections.

In some embodiments, the Ag-specific T cells prepared by the method of the present invention are particularly suitable for the treatment of lymphopenia. Lymphopenia can arise from or be associated with an infection, such as common cold or flu; corticosteroid use; infections with ΗΓ and other viral, bacterial, and fungal agents; malnutrition; systemic lupus erythematosus; severe stress; intense or prolonged physical exercise (due to Cortisol release); rheumatoid arthritis; sarcoidosis; iatrogenic conditions; chemotherapy (such as with cytotoxic agents or immunosuppressive drugs); malignancies such as leukemia or advanced Hodgkin's disease; radiation (large dose (e.g., accidental exposure or whole body radiation)); or post- transplant. In such embodiments, the lymphopenia may be post-transplant lymphopenia, and the Ag-specific T cells may be administered in a donor leukocyte infusion product.

Ag-specific T cells prepared as described above can be utilized in methods and compositions for adoptive immunotherapy in accordance with known techniques, or variations thereof that will be apparent to those skilled in the art based on the instant disclosure. See, e.g., US Patent Application Publication No. 2003/0170238 to Gruenberg et al; see also US Patent No. 4,690,915 to Rosenberg. In some embodiments, the cells are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier) in a treatment-effective amount. Suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized. The infusion medium can be supplemented with human serum albumin. A treatment- effective amount of cells in the composition is dependent on the relative representation of the Ag-specific T cells with the desired specificity, on the age and weight of the recipient, on the severity of the targeted condition and on the immunogenicity of the targeted Ags. These amount of cells can be as low as approximately 10 3 /kg, preferably 5x103 /kg; and as high as

10 7 /kg, preferably 108 /kg. The number of cells will depend upon the ultimate use for which the composition is intended, as will the type of cells included therein. For example, if cells that are specific for a particular Ag are desired, then the population will contain greater than 70%, generally greater than 80%, 85% and 90-95% of such cells. For uses provided herein, the cells are generally in a volume of a liter or less, can be 500 ml or less, even 250 ml or 100 ml or less. The clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed the desired total amount of cells.

As used herein, the term "administering" refers to administration of the compounds as needed to achieve the desired effect. Administration may include, but is not limited to, oral, sublingual, intramuscular, subcutaneous, intravenous, transdermal, topical, parenteral, buccal, rectal, and via injection, inhalation, and implants. As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By a "therapeutically effective amount" is meant a sufficient amount of cells generated with the present invention for the treatment of the disease at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total usage of these cells will be decided by the attending physicians within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and survival rate of the cells employed; the duration of the treatment; drugs used in combination or coincidental with the administered cells; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of cells at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. acDC cytokine cocktails induce equivalent Ag-presenting cells in cord blood and peripheral blood. Peripheral blood mononuclear cells (PBMCs) or cord blood mononuclear cells (CBMCs) were cultured for 48 h with the indicated cytokine cocktails, namely GM-CSF/IL-4, IL-Ιβ, Flt3L or no cytokines during 24 h followed by addition of TNF-a, PGE2, IL-Ιβ and low-dose IL-7 for another 24 h. At the end of this 48 h culture, the phenotype of adherent CDl lc+CD3-CD19- cells was assessed by flow cytometry using the indicated cell surface markers. A. Representative staining obtained from one PBMC (white profiles) and one CBMC sample (grey profiles), as compared to isotype control (dotted line indicating the mean fluorescence intensity, MFI). B. Cumulative data obtained from 3 CBs and 3 PBMC donors, represented as relative MFI + SD for each of the indicated markers, normalized to the MFI registered for PBMC samples in the absence of cytokines.

Figure 2. acDC-stimulated CBMCs efficiently prime MelanA-specific CD8+ T cells once cytokines supporting T-cell survival are added. A. acDC cultures were performed as before in the presence of the indicated cytokines (followed 24 h later by TNF-a, PGE2, IL-Ιβ and low-dose IL-7), either alone (diamonds) or along with IL-15 plus IL-2 (squares) or IL-15 plus IL-2 plus IL-7 (triangles) from day 2 of culture. The stimulation was allowed to proceed for a total of 10 d and the number of total CD8+ T cells obtained counted by flow cytometry using CountBright beads. B. Cultures were performed as above using the GM-CSF/IL-4 acDC cocktail followed by TNF-a, PGE2, IL-Ιβ and low-dose IL-7 along with MelanA27-35 peptide after 24 h and by the indicated homeostatic cytokines from day 2 of culture. The number of MelanA26_35-specific CD8+ T cells obtained at the end of the 10 d culture was counted as above using MelanA26_35-loaded HLA-A2 multimers (MMrs) and absolute numbers were determined with CountBright beads. Results refer to a representative experiment from a single donor out of two tested. Figure 3. acDC-stimulated CBMCs efficiently prime MelanA-and AdV-specific

CD8+ T cells once cytokines supporting T-cell survival are added. HLA-A2+ CBMCs were cultured as above using the indicated acDC cytokine cocktails followed by TNF-a, PGE2, IL-Ιβ and low-dose IL-7 along with MelanA26_35 and a pool of 3 AdV peptides (AdV Hexon542_55o, AdV Hexon892_9oi, AdV Hexon9i6-925) after 24 h and by the indicated homeostatic cytokines from day 2 of culture. The number of MelanA26_35-specific (x-axis; APC fluorochrome) and of AdV-specific CD8+ T cells (y-axis; PE fluorochrome) obtained at the end of the 10 d culture was counted using fluorochrome-labeled HLA-A2 MMrs loaded with the corresponding peptides. Numbers show the absolute counts of MMr+CD8+ T cells, as determined with CountBright beads. Results refer to a representative experiment from a single donor out of three tested.

Figure 4. Variable effects of different acDC cytokine cocktails on the expansion of Ag-specific CD8+ T cells from different CBMC donors. HLA-A2+ CBMCs were cultured as above using the indicated acDC cytokine cocktails followed by TNF-a, PGE2, IL-Ιβ and low-dose IL-7 along with MelanA26-35 and AdV peptides (AdV Hexon542_55o, AdV Hexong92_ 901, AdV Hexon9i6-925) after 24 h and by the indicated cytokines from day 2 of culture. The number of MelanA27_35-specific (x-axis; APC fluorochrome) and of AdV-specific CD8+ T cells (y-axis; PE fluorochrome) obtained at the end of the 10 d culture was counted using fluorochrome-labeled HLA-A2 MMrs loaded with the corresponding peptides. Numbers show the absolute counts of MMr+CD8+ T cells, as determined with CountBright beads. Results refer to a representative experiment from two different donors (top and bottom row, respectively). Figure 5. More complex acDC cytokine cocktails induce Ag-presenting cells of similar phenotype. A. CBMCs (left) and PBMCs (right) were cultured as above using the indicated acDC cytokine cocktails followed by TNF-a, PGE2, IL-Ιβ and low-dose IL-7 after 24 h. At the end of this 48 h culture, the phenotype of adherent CD1 lc+CD3-CD19- cells was assessed by flow cytometry using the indicated cell surface markers. A representative staining obtained from one CBMC (left) and one PBMC sample (right) is shown. B. Cumulative data obtained from 6 different donors (3 CBMC donors, top; and 3 PBMC donors, bottom), represented as the relative representation of CD 14+ and CD 14- cells among adherent CD11C+CD3-CD19- cells expressing 0 to 3 different markers among HLA-DR, CD80 and CD86.

Figure 6. More complex acDC cytokine cocktails induce MelanA27-35-specific CD8+ T cells in similar numbers but of higher polyfunctionality. A. PBMCs and CBMCs (3 donors/each; same donors as in Fig. 5) were cultured as above using the indicated acDC cytokine cocktails followed by TNF-a, PGE2, IL-Ιβ and low-dose IL-7 along with MelanA26- 35 peptide after 24 h and by IL-2, IL-15 and IL-7 from day 2 of culture. Absolute numbers of MelanA26-35-specific CD8+ T cells obtained at the end of the 10 d culture are plotted, as determined with MelanA26_35-loaded HLA-A2 MMrs and CountBright beads. B. The same cultures were tested at day 13 during a 6 h recall assay in the presence of EBV-transformed lymphoblastoid HLA-A2+ LCL cells pulsed with MelanA26_35 or no peptide. The graph displays absolute numbers of CD8+ cells producing at least one cytokine among IFN-γ, TNF- a, IL-2 and MIP-Ιβ in response to MelanA26_35-pulsed LCL cells after background subtraction, i.e. the number of cytokine-positive CD8+ cells detected in response to unpulsed LCL cells. C. Polyfunctionality indexes calculated for the indicated acDC cultures by taking into account the number of cytokines (IFN-γ, TNF-a, IL-2, MIP-Ιβ or none) co-produced by CD8+ T cells.

Figure 7. acDC stimulation in G-Rex devices further increases the yield of Ag- specific CD8+ T cells. A. CBMCs were cultured as above in 48- well plates (2xl06 CBMCs/well) or G-Rex flasks (lOxlO6 CBMCs/flask) using a combination of GM-CSF, IL-4, IL-Ιβ and Flt3L, followed by TLR8L, TNF-a, PGE2 and low-dose IL-7 along with MelanA26_ 35 peptide after 24 h and by IL-2, IL-15 and IL-7 from day 2 of culture. The number of MelanA26_35-specific CD8+ T cells obtained at the end of the 10 d culture is represented after gating on viable CD8+ cells and was counted using MelanA26_35-loaded HLA-A2 MMrs. The absolute numbers of MMr+ cells per million CBMCs obtained are indicated, as determined with CountBright beads. B. The same culture was tested at day 13 during a 6 h recall assay in the presence of LCL cells pulsed with MelanA26-35 or no peptide. The graph displays absolute numbers of CD8+ cells per million CBMCs producing the indicated cytokines in response to MelanA26_35-pulsed LCL cells after background subtraction, i.e. the number of cytokine- positive CD8+ cells detected in response to unpulsed LCL cells. C. Summary of a second experiment performed as above by comparing acDC cultures (GM-CSF, IL-4, IL-Ιβ and FLT3L followed after 24 h by TNF-a, PGE2, TLR8L and low-dose IL-7; and by IL-15, IL-2 and IL-7 after another 24 h) in 24- well plates (5xl06 CBMCs/well) and in G-Rex flasks (lOxlO6 CBMCs/flask). The yields of MelanA27-35- and pooled AdV-specific CD8+ T cells is indicated as number/106 CBMCs and as total number, as determined by MMr staining and CountBright beads. ND, not detectable. D. Summary of a third experiment comparing acDC cultures performed as above in G-Rex flasks (lOxlO6 CBMCs/flask). The yields of total MelanA27_35-, AdV Hexon542_550-, AdV Hexon892_9oi- and AdV Hexon9i6-925-specific CD8+ T cells is indicated, as determined by MMr staining and CountBright beads.

EXAMPLE: Material & Methods

Blood samples. CB units for research purposes were obtained from the CB bank at the Saint Louis Hospital in Paris. Peripheral blood samples were obtained from healthy donors. All donors gave written informed consent and the study was approved by the local Ethics committees. Mononuclear cells were separated by density gradient centrifugation and stored frozen in liquid nitrogen as described (14-16). HLA Class I typing was performed with AmbiSolv primers (Life Technologies). acDC cytokine treatment of peripheral blood and cord blood mononuclear cells. Frozen-thawed peripheral blood mononuclear cells (PBMCs) or cord blood mononuclear cells (CBMCs) were plated at the following densities in AIM-V medium (Life Technologies) depending on the culture vessels used: 2xl06/500 μΐ/well in 48-well plates; 5xl06/l ml/well in 24- well plates; 10xl06/2 ml/well in 12- well plates; and 10xl06/20 ml/flask in G-RexlO flasks (Wilson Wolf). The following cytokines were used for acDC stimulation, added sequentially as detailed for each figure: day 0: GM-CSF (R&D; 1000 U/ml), IL-4 (R&D; 500 U/ml), IL-Ιβ (R&D; 10 ng/ml); Flt3L (R&D; 50 ng/ml); day 1: TLR8L (ssRNA40, Invivogen; 0.5 μg/ml) TNF-a (R&D; 1000 U/ml) PGE2 (Merck Calbiochem; 1 μΜ) and low-dose IL-7 (R&D; 0.5 ng/ml); day 2: IL-2 (Proleukin, Novartis; 100 U/ml); IL-15 (R&D; 25 ng/ml) and IL-7 (R&D; 5 ng/ml), which were added by replacing half medium volume with AIM-V + 10% human serum containing the above cytokines at the indicated final concentrations calculated for the whole culture volume. When IL-Ιβ was added at day 0, it was not further added at day 1. Half medium was replenished every 2-3 days with AEVI-V + 10% human serum, supplemented with 100 U/ml IL-2, 25 ng/ml IL-15 and 5 ng/ml IL-7 when indicated. Phenotyping of Ag-presenting cells. Adherent cells were collected after 48 h of acDC stimulation and phenotype determined using the following antibodies: CD80-FITC (clone BB1), CD86-PE (clone IT2.2), CD14 PerCP-Cy5 (clone M5E2), HLA-DR-APC (clone G46-6), CD3-V450 (clone UCHT1), CD19-V450 (clone HIB 19), all from BD; CDl lc- AlexaFluor700 (clone 3.9; eBioscience) and Live/Dead Aqua (Life Technologies). Cells were acquired using a 16-color BD LSR Fortessa flow cyto meter and analysed with FlowJo software (TreeStar).

Generation of Ag-specific CD8+ T cells by acDC stimulation. CBMCs and PBMCs from HLA-A2+ (HLA-A*02:01+) donors were used and analysed at day 10 of acDC cultures. The following HLA-A2-restricted peptides (synthesized at >85% purity; ChinaPeptides) were added after the first 24 h of acDC culture and used at a 10 μΜ final concentration: Melan-A26- 35 (A27L variant; ELAGIGILTV) (SEQ ID NO: l), AdV5 Hexon542-55o (GLRYRSMLL) (SEQ ID NO:2) (17), AdV5 Hexon892_90i (LLYANSAHAL) (SEQ ID NO:4) (18), AdV5 Hexon916_ 925 (YVLFEVFDVV) (SEQ ID NO:5) (18). HLA-A2 multimers were synthesized using the one-pot, mix-and-read technology (19) and staining performed in the presence of 50 nM dasatinib (20) as described (16). Cells were gated on live (Live/Dead-negative) CD8+ events for analysis. Absolute numbers of MMr+ cells retrieved from each culture were determined with CountBright beads (Life Technologies) following manufacturer's instructions.

Antigen recall assays. HLA-A2+ LCL cells were used as Ag-presenting cells (APCs) and labeled with CFSE (Life Technologies) to separate them from cells retrieved from acDC cultures. They were then pulsed with the indicated peptide at a 10 μΜ final concentration for 2 h. After washing, 0.5xl06 LCL cells were incubated 1: 1 with CBMCs or PBMCs from acDC cultures for 6 h in the presence of 10 μg/ml brefeldin A in 96- well flat-bottom plates. Intracellular cytokine staining (ΜΙΡ-Ιβ-Fluorescein, clone 24006, R&D; IFN-γ-ΡΕ, clone 4S.B3, eBioscience; IL-2-PE-Cy7, clone MQ1-17H12, eBioscience; TNF-a-APC, clone MAbl l, BD) was performed using BD Cytofix/Cytoperm reagents and analyzed on a BD LSR Fortessa cytometer after gating on live CFSE-negative CD8+ events. Polyfunctionality indexes were calculated as previously described (21).

Results

acDC cytokine cocktails induce equivalent Ag-presenting cells in cord blood and peripheral blood.

Cord blood (CB) harbours immune cells with an immature phenotype (22, 23), which are less prone to induce productive immune responses. We therefore asked whether suitable Ag-presenting cells (APCs), could be induced in a CB mononuclear cell (CBMC) mixture, as previously obtained with peripheral blood mononuclear cells (PBMCs) (13), by exposing them for 48 h to different cytokines. Fig. 1A shows a representative staining comparing CBMCs and PBMCs, and Fig. IB show cumulative results obtained from different donors. Exposure to GM-CSF/IL-4, IL-Ιβ or Flt3 ligand (Flt3L) followed by pro-inflammatory cytokines led to identical phenotype changes when comparing CBMCs with PBMCs. The GM-CSF/IL-4 cytokine cocktail led to differentiation of dendritic cells (DCs), as evidenced by CD14 down-regulation and up-regulation of HLA-DR and of the costimulatory molecules CD80 and CD86. Conversely, both IL-Ιβ and Flt3L led to CD 14 up-regulation, without major changes in the expression of HLA-DR, CD80 or CD86, consistent with the induction of different APC populations. Taken together, these results show that acDC protocols can be used to differentiate APCs from both CBMCs and PBMCs with similar results. acDC-stimulated CBMCs efficiently primes Ag-specific CD8+ T cells once cytokines supporting their survival are added, with variable effects of different acDC cytokine cocktails.

CB T cells mostly harbour a naive phenotype and are exquisitely sensitive to apoptosis, a feature that can be corrected by supplementation of homeostatic cytokines such as IL-2, IL-7 and IL-15 (8). We therefore explored the requirement for these common γ chain receptor cytokines to support the survival and expansion of CD8+ T cells. We focused on CD8+ T cells as they are the final effectors of viral clearance, in line with the main therapeutic application envisaged. acDC stimulation during the first 48 h as before was extended for an additional 8 days (i.e. 10 days total), with homeostatic cytokines added from day 2 and replenished every 2-3 days. Previous kinetics studies documented that this 10-day culture represents the optimal time point for maximal retrieval of primed Ag-specific CD8+ T cells (data not shown). As shown in Fig. 2 A, the number of total CD8+ T cells that could be retrieved after these acDC cultures in the presence of different cytokines was significantly increased by the addition of IL-2 and IL-15, alone or in combination with IL-7, with higher yields obtained when IL-7 was included. This was also true when acDC cultures were performed in the presence of an immunodominant MelanA26-35 epitope peptide, here used as a model Ag in light of the high precursor frequencies of cognate CD8+ T cells present in most HLA-A2+ healthy individuals (24). As shown in Fig. 2B, the number of MelanA26_35- specific CD8+ T cells retrieved at day 10 of culture following acDC stimulation with GM-CSF/IL-4 was 17-fold higher in the presence of IL-2/IL-15 or IL-2/IL-15/IL-7. These experiments also showed that a minimal number of starting CBMCs was required for efficient priming of Melan A- specific CD8+ T cells, as no expansion was observed when starting with lxlO6 as compared to 5xl06 CBMCs. This likely reflects the minimal number of T-cell precursors that must be present in the CBMC mixture to be efficiently expanded.

These experiments were then repeated by analysing the expansion obtained with the MelanA26_35 model Ag and with relevant viral epitopes, namely the HLA-A2-restricted AdV Hexon542_55o, AdV Hexon892_9oi and AdV Hexon9i6-925. Also in this case, the expansion was superior once the anti-apoptotic cytokines were included in the cocktail, with better results obtained with IL-15, IL-2 and IL-7 added to the GM-CSF/IL-4 cocktail (Fig. 3). This combination of anti-apoptotic cytokines was therefore retained for further experiments.

While the supporting effect of IL-2, IL-7 and IL-15 was highly reproducible, different acDC cocktails induced expansion of Ag-specific CD8+ T cells with different efficiencies, depending on the targeted Ag and on the CB donor. An example is shown in Fig. 4, in which the expansion of AdV-specific CD8+ T cells was highest with GM-CSF/IL-4 for one donor and with IL-Ιβ for another. Conversely, MelanA26-35- specific CD8+ T-cell expansion was higher for Flt3L in one case, and did not improve as compared to the control culture condition without cytokines in another.

More complex acDC cytokine cocktails induce Ag-presenting cells of similar phenotype and Ag-specific CD8+ T cells in similar numbers but of higher polyfunctionality.

In light of this variability in the expansion yield of Ag-specific CD8+ T cells, a combination of different acDC cytokine cocktails was tested to verify whether a synergistic effect could be achieved, thus maximizing the chances that a given CBMC sample and Ag specificity would respond to such stimulation. First, the phenotype of the APCs obtained with different acDC combinations was studied in parallel in CBMC and PBMC samples, with further inclusion of the TLR8 ligand (TLR8L) sRNA40. A representative example is shown in Fig. 5A and cumulative results in Fig. 5B. First, we observed that IL-4 needed to be combined with GM-CSF to achieve complete CD 14 down-regulation, both in CBMCs and PBMCs. Second, further enrichment of this GM-CSF/IL-4 cocktail with IL-Ιβ and Flt3L, alone or in combination with TLR8L added along with TNF-a, PGE2 and low-dose IL-7 at day 1, did not significantly changed this phenotype, with equivalent CD 14 down-regulation accompanied by similar up-regulation of HLA-DR, CD86 and CD80, both in CBMCs and PBMCs. Cumulative results are visualized in Fig. 5B, where the fractions of CD 14+ and CD 14- cells is represented in greyscale, depending on the number of stimulatory molecules expressed (0, 1, 2 or 3 among HLA-DR, CD80 and CD86), reflecting different degrees of maturation and stimulatory potency. While CD 14+ cells represent cells of the monocyte lineage, the CD 14- fraction represents bona fide DCs. This representation also highlights that, while the APC composition in the absence of IL-4 is mostly made of CD 14+ cells, the CD 14- fraction becomes predominant once IL-4 is added, with a substantial fraction (>50 ) of these cells displaying a mature phenotype expressing 2 to 3 molecules among HLA-DR and co- stimulatory receptors CD80 and CD86. Taken together, these results show that enrichment of the GM-CSF/IL-4 cocktail with additional acDC cytokines does not significantly modify the APC phenotype compared to what obtained with GM-CSF/IL-4 alone.

The number and phenotype of MelanA26_35-specific CD8+ T cells obtained with these different acDC cocktails was then analysed in parallel with APC phenotypes (Fig. 6). Yields were not different when the GM-CSF/IL-4 cocktail was enriched with additional acDC cytokines as above (Fig. 6A). This was true for both PBMCs and CBMCs, with PBMCs yielding higher average numbers than obtained with CBMCs. The number of T cells expressing different cytokines in response to a MelanA26-35 recall was then analysed (Fig. 6B). While the number of cells secreting one or more cytokines among IFN-γ, TNF-a, IL-2 and MIP-Ιβ was similar for PBMCs using GM-CSF/IL-4 either alone or complemented with additional acDC cytokines, cytokine production was higher in CB T cells obtained with acDC cocktails combining GM-CSF/IL-4, IL-Ιβ and Flt3L, with or without addition of TLR8L, as compared to GM-CSF/IL-4 alone. Moreover, the more enriched acDC cytokine cocktail comprising GM-CSF, IL-4, IL-Ιβ, Flt3L and TLR8L led to selection of MelanA26-35-specific CD8+ T cells endowed with higher polyfunctionality (Fig. 6C). This cocktail was therefore retained for further experiments. acDC stimulation in G-Rex devices further increases the yield of Ag-specific CD8+ T cells.

Last, we explored strategies to maximize the yield of Ag-specific T cells obtained during the acDC culture. G-Rex flasks (Wilson Wolf) have been recently described to increase cell culture yields by maximizing gas exchanges from the bottom rather than the top of the vessel, i.e. in closer contact with cells. We therefore compared the expansion obtained in standard culture plates compared to G-Rex flasks, using the acDC cytokine cocktail selected above supplemented with IL-2, IL-7 and IL-15. A representative example is shown in Fig. 7A by using the model Ag MelanA26-35, where acDC stimulation in G-Rex flasks led to an 11 -fold increase in expansion compared to a standard plate culture. Expanded cells were further tested for cytokine production (Fig. 7B), showing consistent higher numbers of TNF- a- and IL-2-producing T cells for G-Rex cultures; and detectable numbers of IFN-γ- and MIP- Ιβ-producing T cells that were not observed in plate cultures. The same comparison was then applied to the expansion of AdV-specific CD8+ T cells, which were stimulated by adding three AdV (Hexon542_55o, Hexong92_9oi, Hexon9i6-92s) and one MelanA26-35 peptides in the same culture (Fig. 7C). Also in this case, Melan A- specific CD8+ T cells were expanded 12- fold more efficiently in G-Rex flasks than in culture plates. This difference was also observed for AdV-specific CD8+ T cells, which displayed lower precursor frequencies. While no expansion was observed when CBMCs were acDC-stimulated in plates, a significant expansion was observed in G-Rex flasks. Another example is shown in Fig. 7D, where the three AdV Hexon epitope- specific CD8+ T-cell fractions were counted separately. The total T-cell yields here obtained starting from a single lOxlO6 frozen-thawed CBMC aliquot for these three AdV specificities were of 3.9xl05 CD8+ T cells. The targeted number of Ag- specific T cells currently used for adoptive transfer is of -5x10 /kg of body weight. Thus, the AdV-specific CD8+ T cells here obtained would be sufficient to treat an average 78-kg individual. Taken together, these data show that coupling of acDC stimulation protocols with suitable culture vessels further enhances the expansion of the viral Ag-specific T-cell fractions of interest to the amounts needed for adoptive T-cell transfer therapies using minimal CBMC numbers.

REFERENCES:

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.

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Claims

CLAIMS:
1. A method for preparing antigen (Ag)-specific T cells from an umbilical cord blood (CB) sample isolated from a subject comprising the steps of i) culturing said CB sample in an appropriate culture medium, which comprises an amount of at least one agent capable of stimulating dendritic cell differentiation and an amount of at least one Ag and ii) isolating said Ag-specific T cells.
2. The method of claim 1 wherein the agent capable of stimulating dendritic cell differentiation is a cytokine.
3. The method of claim 2 wherein the culture medium comprises an amount of Granulocyte/Macrophage Colony-Stimulating Factor (GM-CSF) and an amount of interleukin (IL)-4.
4. The method of claim 2 wherein the culture medium comprises an amount of FMS-like tyrosine kinase 3 (Flt-3) ligand.
5. The method of claim 2 wherein the culture medium comprises an amount of IL-Ιβ.
6. The method of claim 1 wherein the culture medium comprises pro-inflammatory stimuli and/or agents which mimic a viral or bacterial aggression.
7. The method of claim 6 wherein the pro-inflammatory stimuli is selected from the group consisting of tumor necrosis factor alpha (TNF-a), prostaglandin E2 (PGE2), anti-CD40 monoclonal antibodies (mAbs), CD40 ligand (CD40L) recombinant chimeric proteins, interferon-alpha (IFN-a), interferon- gamma (IFN-γ), and IL-7.
8. The method of claim 6 wherein the agent which mimics a viral or bacterial aggression is selected from the group consisting of lipopolysaccharides (LPS), CpG oligodeoxynucleotides, polyinosinic:polycytidylic acid (poly I:C), Pam3CysSerLys4 (Pam3CSK4), imiquimod.
9. The method of claim 1 wherein the agent capable of stimulating dendritic cell differentiation is a ligand suitable for the activation of a pathogen recognition receptor.
10. The method of claim 9 wherein the ligand that is suitable for the activation of a pathogen recognition receptor is a TLR agonist selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR 10, TLR11, TLR 12, or TLR 13 agonists.
11. .The method of claim 1 wherein the culture medium is supplemented with at least one homeostatic cytokine selected from the group consisting of IL-2, IL-7 and IL-15, or combinations thereof.
12. The method of claim 1 wherein the antigen is selected from the group consisting of viral antigens, bacterial antigens, fungal antigens, and cancer-associated antigens.
13. The method of claim 1 which further comprises a step consisting of detecting stimulated T cells.
14. The method of claim 1 which further comprises a step consisting of detecting stimulated T cells.
15. A population of antigen- specific T cells obtainable by the method of claim 1
16. A method of treating cancer, infection, or lymphopenia in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the population of antigen-specific T cells of claim 15.
17. The method of claim 1 wherein the subject undergoes hematopoietic stem cell transplantation using peripheral blood or CB precursors.
18. Use the population of antigen-specific T cells of claim 15 for adoptive immunotherapy.
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