EP3445378A1

EP3445378A1 - Methods and vaccine compositions for the treatment of b-cell malignancies

Info

Publication number: EP3445378A1
Application number: EP17720714.9A
Authority: EP
Inventors: Sylvain LATOUR; Alain Fischer; Emmanuel Martin; Kazushi IZAWA
Original assignee: Assistance Publique - Hôpitaux de Paris; Institut National de la Santé et de la Recherche Médicale; Université Paris Descartes; Fondation Imagine
Current assignee: Assistance Publique - Hôpitaux de Paris; Institut National de la Santé et de la Recherche Médicale; Université Paris Descartes; Fondation Imagine
Priority date: 2016-04-22
Filing date: 2017-04-21
Publication date: 2019-02-27
Also published as: JP2019514870A; WO2017182608A1

Abstract

The present invention relates to methods and vaccine compositions for the treatment of B-cell malignancies. Epstein-Barr virus (EBV) infection in humans is a major trigger of malignant and non-malignant B cell proliferations. The inventors showed that EBV-specific T lymphocytes cannot expand properly when stimulated with CD70-deficient EBV-infected B cells, while expression of CD70 restores expansion. In particular, the present invention relates to a method of treating a B-cell malignancy in a subject in need thereof comprising i) providing a sample of malignant B cells obtained from the subject ii) isolating and culturing a population of malignant B cells from the sample of step i), iii) introducing in the population malignant B cells of step ii) a nucleic acid molecule encoding for a CD70 polypeptide and iv) administering to the subject a therapeutically effective amount of the population of malignant B cells of step iii).

Description

METHODS AND VACCINE COMPOSITIONS FOR THE TREATMENT OF B-CELL

MALIGNANCIES

FIELD OF THE INVENTION:

The present invention relates to methods and vaccine compositions for the treatment of B-cell malignancies.

BACKGROUND OF THE INVENTION:

Epstein-Barr virus (EBV) is a gamma-herpes virus that infects most of humans and has a marked tropism for B lymphocytes. Importantly, EBV is known to be one of the strongest trigger of intrinsically uncontrolled B-cell proliferation and lymphomagenesis. Rare genetic diseases specifically predispose to defective control of EBV infection, leading to virus- associated hemophagocytic syndrome, lymphoproliferative disorders (LPD) such as Hodgkin and non-Hodgkin lymphomas¹' ². At present, mutations in CTPS1, SH2D1A, MAGT1, ITK and CD27 have been associated with high penetrance of EBV infection with up to 90% of patients having developed diseases and lymphomas related to persistent EBV infection³' ⁴' ⁵' ⁶' ⁷' ⁸. Studies of these primary immunodeficiencies uncovered crucial pathways involved in T-cell response towards EBV-infected B lymphocytes and more generally in T-cell functions. In healthy individuals, efficiency of the immune response to EBV is indeed mainly dependent of the massive expansion of specific CD8⁺ cytotoxic T cells that eliminate EBV-infected B cells⁹' ¹⁰. In CTPS1, SH2D1A and MAGT1 deficiencies, CD8⁺ T-cell responses towards EBV-infected B lymphocytes are impaired as the result of defects in either cell-mediated cytotoxicity and/or expansion of specific cytotoxic CD8⁺ T cells. The X-linked lymphoproliferative syndrome (XLP)-l syndrome, characterized by EBV-induced hemophagocytic syndrome and occurrence of B lymphomas, is caused by mutations in SH2D1A coding the signaling lymphocytic activation molecule (SLAM)-associated protein SAP. In XLP-1, the CD8⁺ T cell-cytotoxicity response towards EBV-infected B cells is specifically compromised and abnormal due to impaired activity of SLAM receptors, which depend on SAP for their function¹¹' ¹²' ¹³. MAGT1 codes for a transmembrane Mg²⁺ transporter involved in TCR signaling and expression of NKG2D, an important cytolytic activating cell receptor expressed by CD8⁺ T cells¹⁴' ¹⁵. Thus, the NKG2D and SLAM-SAP pathways represent important components of the immune response to EBV. ITK deficiency is caused by mutations in ITK, a well-characterized tyrosine kinase of the BTK family involved in T-cell activation through its ability to activate the PLC- γΐ ¹⁶. In the absence of ITK, PLC-γΙ activation in response to TCR activation is compromised resulting in decreased Ca²⁺ mobilization¹⁷' ¹⁸. Itk-deficient mice exhibit defective CD8⁺ T-cell expansion during anti- viral responses¹⁹. However, the exact mechanisms underlying the defective immune response to EBV in ITK-deficient patients is not known. A deficiency in CD27 has been recently identified in 17 patients who all developed EBV-driven lymphoproliferative disorders supportive of a key role of CD27 in immunity against EBV, although its exact mechanism of action has not been delineated so far⁵' ⁶' ²⁰' ²¹ ' ²². The ligand of CD27, CD70 is expressed on some B lymphocytes and dendritic cells subpopulations, and transiently found on most immune cells when activated²³. CD70 is also present on a variety of cancer cells including neoplasias of B-cell origin²⁴. CD27 and CD70 are homodimer type I and homotrimer type II membrane proteins, respectively²¹. Both belong to the Tumor Necrosis Factor (TNF) superfamily. Studies of murine models established that the CD27-CD70 pathway plays an important role in the generation and maintenance of T-cell immunity in particular during anti-viral responses²²' ²⁵' ²⁶' ²¹.

SUMMARY OF THE INVENTION:

The present invention relates to methods and vaccine compositions for the treatment of

B-cell malignancies. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Epstein-Barr virus (EBV) infection in humans is a major trigger of malignant and non- malignant B cell proliferations. Inherited ITK and CD27 deficiencies are characterized by impaired immune responses to EBV-infected B cells, though the underlying pathological mechanisms have not yet been identified. Herein, the inventors report a patient suffering from recurrent EBV-induced B cell proliferation due to a deficiency in CD70, the ligand of CD27, a co-stimulatory molecule of T cells. They show that EBV-specific T lymphocytes cannot expand properly when stimulated with CD70-deficient EBV-infected B cells, while expression of CD70 restores expansion. Analysis of ITK-deficient lymphocytes demonstrates that CD70- triggered CD27-mediated T-cell proliferation is fully dependent on ITK that couples CD27 to downstream signaling cascades including calcium mobilization. Thus, the CD70-CD27-ITK pathway appears to be a crucial component of EBV-specific T-cell immunity and more generally for the immune surveillance of B-cells. Accordingly, CD70-CD27 could represent an important target to induce anti-tumoral vaccination. Restoration or induction of CD70 expression in some lymphoma cells lacking CD70 might represent a therapeutic approach to induce a global and potent anti-tumoral immunity.

Accordingly the first object of the present invention relates to a method of treating a B- cell malignancy in a subject in need thereof comprising i) providing a sample of malignant B cells obtained from the subject ii) isolating and culturing a population of malignant B cells from the sample of step i), iii) introducing in the population malignant B cells of step ii) a nucleic acid molecule encoding for a CD70 polypeptide and iv) administering to the subject a therapeutically effective amount of the population of malignant B cells of step iii).

As used herein, the term "B-cell malignancy" includes any type of leukemia or lymphoma of B cells. The term "B cell lymphoma" refers to a cancer that arises in cells of the lymphatic system from B cells. B cells are white blood cells that develop from bone marrow and produce antibodies. They are also known as B lymphocytes. B-cell malignancies include, but are not limited to, non-Hodgkin's lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma, primary effusion lymphoma, diffuse large B-cell lymphoma, splenic marginal zone lymphoma, MALT (mucosa-associated lymphoid tissue) lymphoma, hairy cell leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, B cell lymphomas (e.g. various forms of Hodgkin's disease, B cell non-Hodgkin's lymphoma (NHL) and related lymphomas (e.g. Waldenstrom's macroglobulinaemia (also called lymphoplasmacytic lymphoma or immunocytoma) or central nervous system lymphomas), leukemias (e.g. acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL; also termed B cell chronic lymphocytic leukemia BCLL), hairy cell leukemia and chronic myoblastic leukemia) and myelomas (e.g. multiple myeloma). Additional B cell malignancies include small lymphocytic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, extra-nodal marginal zone B cell lymphoma of mucosa-associated (MALT) lymphoid tissue, nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma/leukemia, grey zone lymphoma, B cell proliferations of uncertain malignant potential, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorder.

In some embodiments, the B-cell lymphoma is caused by Epstein-Barr virus (EBV). In some embodiments, the B cell lymphoma is a diffuse large B-cell lymphoma associated with mutation in the gene encoding for CD70. CD70 is indeed one of the most frequently mutated genes in diffuse large B-cell lymphomas ⁴²' ⁴⁴, ⁴⁵.

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 "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

The sample of malignant B cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, and tumoral tissue. Typically, the B cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FICOLL™ (copolymers of sucrose and epichlorohydrin that may be used to prepare high density solutions) separation. In some embodiments, cells from the circulating blood of an individual are obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated "flow-through" centrifuge (for example, the Cobe 2991 cell processor) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, PBS. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media. B cells may be isolated from peripheral blood or leukapheresis using techniques known in the art. For example, PBMCs may be isolated using FICOLL™ (Sigma-Aldrich, St Louis, Mo.) and CD 19+ B cells purified by negative or positive selection using any of a variety of antibodies known in the art, such as the Rosette tetrameric complex system (StemCell Technologies, Vancouver, Canada). Other isolation kits are commercially available, such as R&D Systems' MagCellect Human B Cell Isolation Kit (Minneapolis, Minn.).

As used herein, the term "CD70" has its general meaning in the art and refers to the ligand for CD27 (see, for example, Bowman M R et al., J. Immunol. 1994 Feb. 15; 152(4):1756- 61). CD70 is also referred to as "CD70 molecule", "CD27L", "CD27LG", "TNFSF7," "tumor necrosis factor (ligand) superfamily member 7," "CD27 ligand," "CD70 antigen," "surface antigen CD70," "tumor necrosis factor ligand superfamily, member 7," "Ki-24 antigen," and "CD27-L". CD70 is a type II transmembrane protein that belongs to the tumor necrosis factor (TNF) ligand family. It is a surface antigen on activated T and B lymphocytes that induces proliferation of co-stimulated T cells, enhances the generation of cytolytic T cells, and contributes to T cell activation. It has also been suggested that CD70 plays a role in regulating B-cell activation, cytotoxic function of natural killer cells, and immunoglobulin synthesis (Hintzen R Q et al., J. Immunol. 1994 Feb. 15; 152(4): 1762-73). An exemplary human amino acid sequence of CD70 is reference in GenBank under the Accession No. NP— 001243 (SEQ ID NO: 1):

SEQ ID NO: 1

MPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPLESL GWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIY MVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTP LARGDTLCTN LTGTLLPSRNTDETFFGVQWVRP

In some embodiments, the malignant B cells are transformed with a nucleic acid molecule encoding for a polypeptide comprising an amino acid sequence having at least 90% of identity with SEQ ID NO: l

According to the invention a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar are the two sequences. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math., 2:482, 1981; Needleman and Wunsch, J. Mol. Biol, 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A., 85:2444, 1988; Higgins and Sharp, Gene, 73:237-244, 1988; Higgins and Sharp, CABIOS, 5: 151-153, 1989; Corpet et al. Nuc. Acids Res., 16: 10881-10890, 1988; Huang et al., Comp. Appls Biosci., 8: 155- 165, 1992; and Pearson et al, Meth. Mol. Biol, 24:307-31, 1994). Altschul et al, Nat. Genet., 6:119-129, 1994, presents a detailed consideration of sequence alignment methods and homology calculations. By way of example, the alignment tools ALIGN (Myers and Miller, CABIOS 4: 11-17, 1989) or LFASTA (Pearson and Lipman, 1988) may be used to perform sequence comparisons (Internet Program® 1996, W. R. Pearson and the University of Virginia, fasta20u63 version 2.0u63, release date December 1996). ALIGN compares entire sequences against one another, while LFASTA compares regions of local similarity. These alignment tools and their respective tutorials are available on the Internet at the NCSA Website, for instance. Alternatively, for comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function can be employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). The BLAST sequence comparison system is available, for instance, from the NCBI web site; see also Altschul et al, J. Mol. Biol, 215:403-410, 1990; Gish. & States, Nature Genet., 3:266-272, 1993; Madden et al. Meth. EnzymoL, 266: 131-141, 1996; Altschul et al, Nucleic Acids Res., 25:3389-3402, 1997; and Zhang & Madden, Genome Res., 7:649-656, 1997.

As used herein, the term "nucleic acid molecule" has its general meaning in the art and refers to a DNA or RNA molecule. However, the term captures sequences that include any of the known base analogues of DNA and RNA such as, but not limited to 4-acetylcytosine, 8- hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-

(carboxyhydroxylmethyl) uracil, 5-fiuorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5 -carboxymethyl-aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1 -methylpseudouracil, 1-methylguanine, 1- methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil- 5-oxyacetic acid methylester, uracil-5 -oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, -uracil-5 - oxyacetic acid methylester, uracil-5 -oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

As used herein, the term "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA has been "transformed".

Typically, the transformation of the malignant B cells with the nucleic acid molecule employs viral vectors to transduce B cells. Examples of viral vectors include, without limitation, adenovirus-based vectors, adeno-associated virus (AAV)-based vectors, retroviral vectors, retroviral-adenoviral vectors, and vectors derived from herpes simplex viruses (HSVs), including amplicon vectors, replication-defective HSV and attenuated HSV (see, e.g., Krisky, Gene Ther. 5: 1517-30, 1998; Pfeifer, Annu. Rev. Genomics Hum. Genet. 2: 177-211, 2001, each of which is incorporated by reference in its entirety). In some embodiments, the B cell are transduced with retroviral vectors, or vectors derived from retroviruses. "Retroviruses" are enveloped RNA viruses that are capable of infecting animal cells, and that utilize the enzyme reverse transcriptase in the early stages of infection to generate a DNA copy from their RNA genome, which is then typically integrated into the host genome. Examples of retroviral vectors Moloney murine leukemia virus (MLV)-derived vectors, retroviral vectors based on a Murine Stem Cell Virus, which provides long-term stable expression in target cells such as hematopoietic precursor cells and their differentiated progeny (see, e.g., Hawley et al, PNAS USA 93: 10297-10302, 1996; Keller et al, Blood 92:877-887, 1998), hybrid vectors (see, e.g., Choi, et al, Stem Cells 19:236-246, 2001), and complex retrovirus-derived vectors, such as lentiviral vectors. As noted above, in some embodiments employ lentiviral vectors. The term "lentivirus" refers to a genus of complex retroviruses that are capable of infecting both dividing and non-dividing cells. Examples of lentiviruses include HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2), visna-maedi, the caprine arthritis-encephalitis virus, equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immune deficiency virus (BIV), and simian immunodeficiency virus (SIV). Lentiviral vectors can be derived from any one or more of these lentiviruses (see, e.g., Evans et al, Hum Gene Ther. 10: 1479-1489, 1999; Case et al, PNAS USA 96:2988-2993, 1999; Uchida et al, PNAS USA 95 : 1 1939-1 1944, 1998; Miyoshi et al, Science 283 :682-686, 1999; Sutton et al, J Virol 72:5781-5788, 1998; and Frecha et al, Blood. 1 12:4843-52, 2008, each of which is incorporated by reference in its entirety). In some embodiments the retroviral vector comprises certain minimal sequences from a lentivirus genome, such as the HIV genome or the SIV genome. The genome of a lentivirus is typically organized into a 5 ' long terminal repeat (LTR) region, the gag gene, the pol gene, the env gene, the accessory genes (e.g., nef, vif, vpr, vpu, tat, rev) and a 3 ' LTR region. The viral LTR is divided into three regions referred to as U3, R (repeat) and U5. The U3 region contains the enhancer and promoter elements, the U5 region contains the polyadenylation signals, and the R region separates the U3 and U5 regions. The transcribed sequences of the R region appear at both the 5 ' and 3 ' ends of the viral RNA (see, e.g., "RNA Viruses: A Practical Approach" (Alan J. Cann, Ed., Oxford University Press, 2000); O Narayan, J. Gen. Virology. 70: 1617-1639, 1989; Fields et al, Fundamental Virology Raven Press., 1990; Miyoshi et al, J Virol. 72:8150-7, 1998; and U.S. Pat. No. 6,013,516, each of which is incorporated by reference in its entirety). Lentiviral vectors may comprise any one or more of these elements of the lentiviral genome, to regulate the activity of the vector as desired, or, they may contain deletions, insertions, substitutions, or mutations in one or more of these elements, such as to reduce the pathological effects of lentiviral replication, or to limit the lentiviral vector to a single round of infection. Typically, a minimal retroviral vector comprises certain 5^f LTR and 3' LTR sequences, one or more genes of interest (to be expressed in the target cell), one or more promoters, and a cis-acting sequence for packaging of the RNA. Other regulatory sequences can be included, as described herein and known in the art. The viral vector is typically cloned into a plasmid that may be transfected into a packaging cell line, such as a eukaryotic cell (e.g., 293-HEK), and also typically comprises sequences useful for replication of the plasmid in bacteria. Typically, the nucleic acid molecule of t interest is located between the 5^f LTR and 3' LTR sequences. Further, the nucleic acid molecule of interest is preferably in a functional relationship with other genetic elements, for example, transcription regulatory sequences such as promoters and/or enhancers, to regulate expression of the gene of interest in a particular manner once the gene is incorporated into the target cell. In some embodiments, the useful transcriptional regulatory sequences are those that are highly regulated with respect to activity, both temporally and spatially. In some embodiments, the viral vectors such as retroviral vectors employ one or more heterologous promoters, enhancers, or both. In some embodiments, the U3 sequence from a retroviral or lentiviral 5 ' LTR may be replaced with a promoter or enhancer sequence in the viral construct. In some embodiments employ an "internal" promoter/enhancer that is located between the 5' LTR and 3' LTR sequences of the viral vector, and is operably linked to the gene of interest. A "functional relationship" and "operably linked" mean, without limitation, that the gene is in the correct location and orientation with respect to the promoter and/or enhancer, such that expression of the gene will be affected when the promoter and/or enhancer is contacted with the appropriate regulatory molecules. Any enhancer/promoter combination may be used that either regulates (e.g., increases, decreases) expression of the viral RNA genome in the packaging cell line, regulates expression of the selected gene of interest in an infected target cell, or both. A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoters are untranslated sequences that are located upstream (5 ' ) of the start codon of a selected gene of interest (typically within about 100 to 1000 bp) and control the transcription and translation of the coding polynucleotide sequence to which they are operably linked. Promoters may be inducible or constitutive. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as a change in temperature. A variety of promoters are known in the art, as are methods for operably linking the promoter to the polynucleotide coding sequence. Both native promoter sequences and many heterologous promoters may be used to direct expression of the selected gene of interest. In some embodiments, the promoter is a heterologous promoters, because they generally permit greater transcription and higher yields of the desired protein as compared to the native promoter. In some embodiments the promoter is selected among heterologous viral promoters. Examples of such promoters include those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus, bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40). In some embodiments the promoter is a heterologous mammalian promoter, such as the actin promoter, an immunoglobulin promoter, a heat-shock promoter, or a promoter that is associated with the native sequence of the gene of interest. Typically, the promoter is compatible with the target cell, such as a quiescent B-lymphocyte, an activated B-lymphocyte, a plasma B cell, or a memory B cell. Non- limiting examples of constitutive promoters that may be used include the promoter for ubiquitin, the CMV promoter (see, e.g., Karasuyama et al, J. Exp. Med. 169: 13, 1989), the β-actin (see, e.g., Gunning et al, PNAS USA 84:4831-4835, 1987), and the pgk promoter (see, e.g., Adra et al, Gene 60:65-74, 1987); Singer-Sam et al, Gene 32:409-417, 1984; and Dobson et al, Nucleic Acids Res. 10:2635-2637, 1982, each of which is incorporated by reference). Non- limiting examples of tissue specific promoters include the Ick promoter (see, e.g., Garvin et al, Mol. Cell Biol. 8:3058-3064, 1988; and Takadera et al, Mol. Cell Biol. 9:2173-2180, 1989), the myogenin promoter (Yee et al., Genes and Development 7: 1277-1289. 1993), and the thyl (see, e.g., Gundersen et al, Gene 113:207-214, 1992). Additional examples of promoters include the ubiquitin-C promoter, the human μ heavy chain promoter or the Ig heavy chain promoter (e.g., MH-M2), and the human κ light chain promoter or the Ig light chain promoter (e.g., EEK-M2), which are functional in B-lymphocytes. In some embodiments, promoters may be selected to allow for inducible expression of the gene. A number of systems for inducible expression are known in the art, including the tetracycline responsive system and the lac operator-repressor system. It is also contemplated that a combination of promoters may be used to obtain the desired expression of the gene of interest. The skilled artisan will be able to select a promoter based on the desired expression pattern of the gene in the organism and/or the target cell of interest.

In some embodiments, the viral vectors (e.g., retroviral, lentiviral) provided herein are "pseudo-typed" with one or more selected viral glycoproteins or envelope proteins, mainly to target selected cell types. Pseudo-typing refers to generally to the incorporation of one or more heterologous viral glycoproteins onto the cell-surface virus particle, often allowing the virus particle to infect a selected cell that differs from its normal target cells. A "heterologous" element is derived from a virus other than the virus from which the RNA genome of the viral vector is derived. Typically, the glycoprotein-coding regions of the viral vector have been genetically altered such as by deletion to prevent expression of its own glycoprotein. Merely by way of illustration, the envelope glycoproteins gp41 and/or gpl20 from an HIV-derived lentiviral vector are typically deleted prior to pseudo-typing with a heterologous viral glycoprotein. In some embodiments, the viral vector is pseudo-typed with a heterologous viral glycoprotein that targets plasma cells such as B-lymphocytes. In some embodiments, the viral glycoprotein allows selective infection or transduction of resting or quiescent B-lymphocytes. In some embodiments, the viral glycoprotein allows selective infection of activated B- lymphocytes. In some embodiments, the viral glycoprotein allows infection or transduction of both quiescent B-lymphocytes and activated B-lymphocytes. In some embodiments, viral glycoprotein allows infection of B cell chronic lymphocyte leukemia cells. In some embodiments, the heterologous viral glycoprotein is derived from the glycoprotein of the measles virus, such as the Edmonton measles virus. In some embodiments pseudo-type the measles virus glycoproteins hemagglutinin (H), fusion protein (F), or both (see, e.g., Frecha et al, Blood. 112:4843-52, 2008; and Frecha et al, Blood. 114:3173-80, 2009, each of which is incorporated by reference in its entirety). In some embodiments, the viral vector comprises an embedded antibody binding domain, such as one or more variable regions (e.g., heavy and light chain variable regions) which serves to target the vector to a particular cell type.

Generation of viral vectors can be accomplished using any suitable genetic engineering techniques known in the art, including, without limitation, the standard techniques of restriction endonuclease digestion, ligation, transformation, plasmid purification, PCR amplification, and DNA sequencing, for example as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y. (1989)), Coffin et al. (Retroviruses. Cold Spring Harbor Laboratory Press, N.Y. (1997)) and "RNA Viruses: A Practical Approach" (Alan J. Cann, Ed., Oxford University Press, (2000)).

Typically, the B cells may be transduced with the viral vectors described herein using any of a variety of known techniques in the art (see, e.g., Science 12 Apr. 1996 272: 263-267; Blood 2007, 99:2342-2350; Blood 2009, 113: 1422-1431; Blood 2009 Oct. 8; 114(15):3173-80; Blood. 2003; 101(6) :2167-2174; Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, N.Y. (2009)). For example, PBMCs, B- or T- lymphocytes from donors and other B cell cancer cells such as B-CLLs may be isolated and cultured in RPMI 1640 (GibcoBRL Invitrogen, Auckland, New Zealand) or other suitable medium, either serum- free or supplemented with 10% FCS and penicillin/streptomycin and/or other suitable supplements. In some embodiments, cells are seeded at 10⁵ cells in 48-wellplates and concentrated vector added at various doses that may be routinely optimized by the skilled person using routine methodologies. In some embodiments, B cells may be transferred to MS5 cell monolayer in RPMI supplemented with 10% AB serum, 5% FCS, 50 ng/ml rhSCF, 10 ng/ml rhIL-15 and 5 ng/ml rhIL-2 and medium refreshed periodically as needed. As would be recognized by the skilled person, other suitable media and supplements may be used as desired.

In some embodiments, the B cells are contacted with a retroviral vector as described herein comprising a nucleic acid of interest operably linked to a promoter, under conditions sufficient to transduce at least a portion of the B cells. In some embodiments the B cells are contacted with a retroviral vector as described herein comprising a nucleic acid of interest operably linked to a promoter, under conditions sufficient to transduce at least 20% of the B cells. In some embodiments, the B cells are contacted with a vector as described herein under conditions sufficient to transduce at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even 100% of the B cells. Any of a variety of culture media may be used in the present methods as would be known to the skilled person (see e.g., Current Protocols in Cell Culture, 2000-2009 by John Wiley & Sons, Inc.). In some embodiments, media for use in the methods described herein includes, but is not limited to Iscove modified Dulbecco medium (with or without fetal bovine or other appropriate serum). Illustrative media also includes, but is not limited to, RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20. In some embodiments, the medium may comprise a surfactant, an antibody, plasmanate or a reducing agent (e.g. N-acetyl-cysteine, 2-mercaptoethanol), or one or more antibiotics. In some embodiments, IL-6, soluble CD40L, and a cross-linking enhancer may also used.

In some embodiments, cells are cultured for 1-7 days. In some embodiments, cells are cultured 7, 14, 21 days or longer. Thus, cells may be cultured under appropriate conditions for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or more days. Cells are replated, media and supplements may be added or changed as needed using techniques known in the art. In some embodiments, the transduced B cells may be cultured under conditions and for sufficient time periods such that at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%), 98%o, 99%) or 100% of the cells express the transgene of interest.

The population of malignant B cells transformed with the nucleic acid molecule encoding for the CD70 polypeptide is thus particularly suitable for inducting anti-tumoral vaccination. In particular, said population of cells will promote the T-cell mediated immunity against tumor cells.

The population of malignant cells transformed according to the invention may be administered either alone, or as a vaccine composition in combination with diluents and/or with other components such as cytokines or cell populations. Briefly, the vaccine compositions of the present invention the population of transformed malignant B cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present invention are preferably formulated for intravenous administration. The vaccine compositions of the present invention may be administered in a manner appropriate to the B cell malignancy to be treated. The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's malignancy, although appropriate dosages may be determined by clinical trials. When "a therapeutically effective amount", "an anti-tumor effective amount", "a tumor-inhibiting effective amount", or "therapeutic amount" is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a vaccine composition comprising the B cells described herein may be administered at a dosage of 10⁴ to 10⁷ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg body weight, including all integer values within those ranges. Vaccine compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al, New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly. Typically, in related adoptive immunotherapy studies, antigen-specific T cells are administered approximately at 2>< 10⁹ to 2x lOⁿ cells to the patient. (See, e.g., U.S. Pat. No. 5,057,423). In some embodiments, lower numbers of the transduced B cells of the present invention, in the range of 10⁶/kilogram (10⁶-10^u per patient) may be administered. In some embodiments, the B cells are administered at 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, or 10¹² cells to the subject. The vaccine compositions may be administered multiple times at dosages within these ranges. The administration of the subject of the vaccine compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In some embodiments, the vaccine compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In some embodiments, the vaccine compositions as described herein are preferably administered by i.v. injection. The vaccine composition may be injected directly into a tumor, or lymph node.

In some embodiments, the vaccine composition is administered to the subject in conjunction with (e.g. before, simultaneously or following) any number of relevant treatment modalities, including but not limited to treatment with agents such as antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin). (Liu et al, Cell 66:807-815, 1991; Henderson et al, Immun. 73:316-321, 1991; Bierer et al, Curr. Opin. Immun. 5:763-773, 1993; Isoniemi (supra)). In some embodiments, the vaccine compositions of the present invention are administered to a patient in conjunction with (e.g. before, simultaneously or following) bone marrow transplantation, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In some embodiments, the vaccine compositions of the present invention are administered following B- cell ablative therapy such as agents that react with CD20. Examples of antibodies having specificity for CD20 include: "C2B8" which is now called "Rituximab" ("RITUXAN®") (U.S. Pat. No. 5,736,137, expressly incorporated herein by reference), a chimaeric pan-B antibody targeting CD20; the yttrium-[90]-labeled 2B8 murine antibody designated "Y2B8" or "Ibritumomab Tiuxetan" ZEVALIN® (U.S. Pat. No. 5,736,137, expressly incorporated herein by reference), a murine IgGl kappa mAb covalently linked to MX-DTPA for chelating to yttrium-[90]; murine IgG2a "BI," also called "Tositumomab," optionally labeled with radioactive 1311 to generate the "1311-B1" antibody (iodine 131 tositumomab, BEXXAR™) (U.S. Pat. No. 5,595,721, expressly incorporated herein by reference); murine monoclonal antibody "1F5" (Press et al. Blood 69 (2):584-591 (1987) and variants thereof including "framework patched" or humanized 1F5 (WO03/002607, Leung, S.; ATCC deposit HB- 96450); murine 2H7 and chimeric 2H7 antibody (U.S. Pat. No. 5,677,180, expressly incorporated herein by reference); humanized 2H7, also known as ocrelizumab (PRO-70769); Ofatumumab (Arzerra), a fully human IgGl against a novel epitope on CD20 huMax-CD20 (Genmab, Denmark; WO2004/035607 (U.S. Ser. No. 10/687,799, expressly incorporated herein by reference)); AME-133 (ocaratuzumab; Applied Molecular Evolution), a a fully- humanized and optimized IgGl mAb against CD20; A20 antibody or variants thereof such as chimeric or humanized A20 antibody (cA20, bA20, respectively) (U.S. Ser. No. 10/366,709, expressly incorporated herein by reference, Immunomedics); and monoclonal antibodies L27, G28-2, 93-1B3, B-CI or NU-B2 available from the International Leukocyte Typing Workshop (Valentine et al, In: Leukocyte Typing III (McMichael, Ed., p. 440, Oxford University Press (1987)). Further, suitable antibodies include e.g. antibody GAlOl (obinutuzumab), a third generation humanized anti-CD20-antibody of Biogen Idec/Genentech/Roche. Moreover, BLX- 301 of Biolex Therapeutics, a humanized anti CD20 with optimized glycosylation or Veltuzumab (bA20), a 2nd-generation humanized antibody specific for CD20 of Immunomedics or DXL625, derivatives of veltuzumab, such as the bispecific hexavalent antibodies of IBC Pharmaceuticals (Immunomedics) which are comprised of a divalent anti- CD20 IgG of veltuzumab and a pair of stabilized dimers of Fab derived from milatuzumab, an anti-CD20 mAb enhanced with InNexus' Dynamic Cross Linking technology, of Inexus Biotechnology both are humanized anti-CD20 antibodies are suitable. Further suitable antibodies are BM-ca (a humanized antibody specific for CD20 (Int J. Oncol. 2011 February; 38(2):335-44)), C2H7 (a chimeric antibody specific for CD20 (Mol Immunol. 2008 May; 45(10):2861-8)), PR0131921 (a third generation antibody specific for CD20 developed by Genentech), Reditux (a biosimilar version of rituximab developed by Dr Reddy's), PBO-326 (a biosimilar version of rituximab developed by Probiomed), a biosimilar version of rituximab developed by Zenotech, TL-011 (a biosimilar version of rituximab developed by Teva), CMAB304 (a biosimilar version of rituximab developed by Shanghai CP Guojian), GP-2013 (a biosimilar version of rituximab developed by Sandoz (Novartis)), SAIT-101 (a biosimilar version of rituximab developed by Samsung BioLogics), a biosimilar version of rituximab developed by Intas Biopharmaceuticals, CT-P10), a biosimilar version of rituximab developed by Celltrion), a biosimilar version of rituximab developed by Biocad, Ublituximab (LFB-R603, a transgenically produced mAb targeting CD20 developed by GTC Biotherapeutics (LFB Biotechnologies)), PF-05280586 (presumed to be a biosimilar version of rituximab developed by Pfizer), Lymphomun (Bi-20, a trifunctional anti-CD20 and anti-CD3 antibody, developed by Trion Pharma), a biosimilar version of rituximab developed by Natco Pharma, a biosimilar version of rituximab developed by iBio, a biosimilar version of rituximab developed by Gedeon Richter/Stada, a biosimilar version of rituximab developed by Curaxys, a biosimilar version of rituximab developed by Coherus Biosciences/Daiichi Sankyo, a biosimilar version of rituximab developed by BioXpress, BT-D004 (a biosimilar version of rituximab developed by Protheon), AP-052 (a biosimilar version of rituximab developed by Aprogen), a biosimilar version of ofatumumab developed by BioXpress, MG-1106 (a biosimilar version of rituximab developed by Green Cross), IBI-301 (a humanized monoclonal antibody against CD20 developed by Innovent Biologies), BVX-20 (a humanized mAb against the CD20 developed by Vaccinex), 20-C2-2b (a bispecific mAb-IFNalpha that targets CD20 and human leukocyte antigen-DR (HLA-DR) developed by Immunomedics), MEDI-552 (developed by Medlmmune/AstraZeneca), the anti-CD20/streptavidin conjugates developed by NeoRx (now Poniard Pharmaceuticals), the 2nd generation anti-CD20 human antibodies developed by Favrille (now MMRGlobal), TRU-015, an antibody specific for CD20 fragment developed by Trubion/Emergent BioSolutions, as well as other precloinical approaches by various companies and entities.

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. Decreased cytolytic and proliferative responses to autologous CD70- deficient LCLs of the patient are restored by expression of CD70 in LCLs.

(a) Cytotoxic response of T cells from two control individuals (Ctr. l and Ctr.2) and the CD70-deficient patient (Pat.) against autologous LCLs as measured by Cr⁵¹ release at the indicated effector-to-target (E:T) ratios. T cells have been co cultured with the autologous LCLs for 4 weeks before the test. One representative of two independent experiments. Triplicates with s.d.

(b) FACS histograms of CD70 expression on LCLs of the patient that have been infected with an empty lentiviral vector (empty vector) or a vector containing a cDNA coding wild-type

CD70 (pLenti vector). One representative of five independent experiments.

(c) Same as (a) except that T cells from the patient were co-cultured with autologous LCLs infected with an empty lentiviral vector (empty vector) or a vector containing a cDNA coding wild-type CD70 (pLenti-CD70). Duplicates with s.d.

(d) Proliferation of T cells from PBMCs of the patient, HLA-A matched (Ctrl.1 HLA-

A01 *) or non-matched (Ctr.2 HLA-A02* and Ctr.3 HLA-A03*) healthy controls that were co cultured for 8 days in the presence of irradiated CD70-deficient LCLs (empty vector) or LCLs in which CD70 expression was restored (pLenti-CD70). Representative dot plots of violet dye dilution and CD25 expression gated on CD3⁺ cells. One representative of five independent experiments.

Figure 2: Analysis of cytolytic activity of T cells expanded from PBMCs of one healthy control (Ctr.) and the CD70-deficient patient (Pat.) for 8-15 days with irradiated autologous CD70-expressing (empty CRISPR or pLenti-CD70) or CD70-deficient (CD70- CRISPR or empty vector) LCL cells. Cytolytic activity of T cells was then tested against a mixture of autologous CD70-expressing (in blue) or CD70-deficient (in red) LCL cells as target cells at a ratio effector-to-target 1 : 1 of 1 for 0, 4 and 12 hours. Residual target cells were evaluated by FACS analysis. Data are represented in percentages of cells normalized to the percentages at time 0.

EXAMPLE: Material & Methods

Ethics. Informed consent was obtained from donors, patients and families of patients. The study and protocols are conform to the 1975 declaration of Helsinki as well as to local legislation and ethical guidelines from the Comite de Protection des Personnes de l'lle de France II, Hopital Necker-Enfants Malade, Paris.

Patients. Individuals with infectious mononucleosis were diagnosed on tonsil biopsies and confirmed with anti-VCA and anti-EBNA IgM positive serology. The main clinical phenotype of patients with ITK and CD27 deficiencies was EBV-driven lymphoproliferation. The patient carrier of the homozygous c.113T>C, p.L38P in ITK is issued from consanguineous parents of Kurdish origin and suffered from recurrent respiratory tract infections with low immunoglobulins levels and developed an EBV-associated lymphoma. Her younger brother and a maternal uncle carried the same deleterious allele and displayed the same clinical phenotype. The two patients carriers of the homozygous c.85C>T, p.R29C in ITK are issued from consanguineous parents of Algerian origin. ITK gene defects were identified by WES and identified mutations were further verified by direct DNA sequencing. The patient carrier of the homozygous c.329G>A, p.Wl 10X in CD27 is issued from consanguineous parents of Tunisian origin and suffered from a recurrent EBV-driven lymphoproliferative disease. The CD27 defect was diagnosed based on the lack of CD27 expression on B cells. The four exons of CD27 were further sequenced.

Exome sequencing and analysis. Exome capture was performed according to the manufacturer's protocol using the Illumina TruSeq exome enrichment kit and sequencing of 100 bp paired end reads on an Illumina HiSeq. Approximately 10 Gb of sequence were obtained for each subject such that 90% of the coding bases of the exome defined by the consensus coding sequence (CCDS) project were covered by at least 10 reads. Adaptor sequences and quality trimmed reads were removed using the Fastx toolkit (http ://hannonlab . cshl.edu/fastx_toolkit/) and a custom script was then used to ensure that only read pairs with both mates present were subsequently used. Reads were aligned to hgl9 with BWA31, and duplicate reads were marked using Pi card (http://picard.sourceforge.net/) and excluded from downstream analyses. Single nucleotide variants (SNVs) and short insertions and deletions (indels) were determined using samtools (http://samtools.sourceforge.net/) pileup and varFilter32 with the base alignment quality (BAQ) adjustment disabled, they were then quality filtered to require at least 20% of reads supporting the variant call. Variants were annotated using both A NOVAR33 and custom scripts to identify whether they affected protein coding sequences, and whether they had previously been seen in the public data bases of exomes and the 7566 exomes previously sequenced at our center. The CD70 variation identified in the patient (19:6586078G/A), a homozygous nonsense mutation c.535C>T p.Argl79X was present the database of the exome aggregation consortium (ExAC) (http://exac.broadinstitute.org) and in our institute database as a heterozygous mutation with a frequency of 1.48.10^"5 (2/134 542 allele). It was not reported in other available public databases of exomes (dbSNP, the 1000 Genomes, the NHLBI Exome Sequencing Project (http://evs.gs.washington.edu/EVS/).

DNA sequencing. Genomic DNA from peripheral blood cells of the patient, their parents, and other family members was isolated according to standard methods. Oligonucleotide primers in the introns flanking the exon 3 of CD70, the exon 1 oflTK and the exon 3 of CD27 were used to amplify genomic DNA. PCR products were amplified using high fidelity Platinum Taq DNA Polymerase (Invitrogen) according to the manufacturer's recommendations, purified with the QIAquick gel extraction kit (Qiagen), sequenced using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (PerkinElmer) according to the manufacturer's recommendations and analyzed with 3500xL Genetic Analyzer (Applied Biosystems). All collected sequences were analyzed using 4peaks software (Version 1.7.2; A. Griekspoor and T. Groothuis, http://mekentosj.com/4peaks/) or DNADynamo (BlueTractorSoftware).

Immunochemistry. Staining and in situ hybridization were performed on a Leica Bond Max automated stainer (Leica Biosystems). The presence of EBV was demonstrated by in situ hybridization for the small RNA encoding region 1 and 2 (EBER). Antibodies used and dilution: anti-CD70 (rabbit polyclonal, 1/200 from Abgent), anti-PAX5 (clone DAK-PAX5, 1/30 from DAKO), anti-CD27 (clone 137B4, 1/50 from ABCAM), anti-CD8 (clone C8/144B, 1/200, from DAKO) and anti-LMPl (clone CS.1-4, 1/200, from DAKO).

Cell culture. Whole blood samples were collected from the patient and control donors. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque density gradient (Lymphoprep, Proteogenix) from blood samples using standard procedures. Expansion of T- cell blasts were obtained by incubating PBMCs for 72 h with phytohaemagglutinin (PHA) (2.5 μg ml^"1, Sigma-Aldrich) in Panserin 401 (Pan Biotech) supplemented with 5% human male AB serum (BioWest), penicillin (100 U ml^"1) and streptomycin (100 μg ml^"1). After 3 days, dead cells were removed by Ficoll-Paque density gradient and blasts were maintained in culture with IL-2 (100 or 1000 UI ml^"1). The Burkitt's lymphoma cell line Raji and the mouse thymoma L1210 obtained from ATCC were cultured in RPMI 1640 GlutaMax medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (Gibco), penicillin (100U ml^"1) and streptomycin (100μ§ ml^"1). Cells were free of mycoplasma and tested for mycoplasma contamination on a regular basis.

EBV-transformed LCLs and EBV-specific T cell lines. EBV-transformed lymphoblastoid cell lines (LCLs) were generated from fresh or frozen PBMCs of the patient and control donors. The PBMCs were incubated with supernatant from B95-8 cells in the presence of ^g/ml of cyclosporine A (Sigma- Aldrich) as previously described⁴⁹. EBV-specific T cell lines were generated from the patient and control healthy donors. PBMCs were co- cultured with 40 Gy irradiated autologous LCLs at an effector-to-stimulator (E/S) ratio of 40: 1. After 9-14 days, viable cells were stimulated with 40 Gy irradiated autologous LCLs at E/S ratio 4: 1 for 5-7days. And then analyzed for cytotoxicity.

Gene expression analysis. Total mRNA was extracted from EBV-B cell lines and activated T-cell lines of the patient and control donors with Pure Link RNA Mini kit (Life technologies) and treated with DNase I. Reverse transcription was performed with Superscript II first-strand synthesis system (Life technologies). Real-time quantitative PCRs for CD70 and Actin were performed in triplicate using ViiA 7 Real-Time PCR system (Life Technologies), SYBR Select Master Mix (Life technologies).

Cell-cytotoxicity assays. The cytolytic activity of CTLs was evaluated with a standard 4-h Cr⁵¹ release assay. In brief, l x lO⁵ cells target cells labeled with Cr⁵¹ were incubated with effector cells for 4 hours at 37 °C at the indicated effector-to-target (E:T) ratios. 30μ1 of supernatant was then collected and counted in with a MicroBetaTrilux and JET counter (PerkinElmer) with 180μ1 of OpitPhase supermix cocktail (Perkin Elmer). Specific lysis (%) was calculated according to the formula: 100 x (experimental release - spontaneous release) / (maximum release - spontaneous release). Cytolytic activity of T cells expanded for 8 days from PBMCs was also evaluated in co cultures against autologous cell trace violet-labeled CD70-expressing LCLs and CFSE-labeled CD70-deficient LCLs, as at a ratio effector-to-target of 1 :1 for 0, 4 and 12 hours. Residual target cells were evaluated by FACS analysis. Data are represented in percentages of cells normalized to the percentages at time 0. Degranulation of CD8⁺ T cells was determined by analysis of the expression of CD107/LAMP, a marker of the exocytosis of lytic granules as described before ⁸.

Stimulation and proliferation assays. PHA-stimulated T cells were washed and cultured without IL-2 for 72 hours to synchronize the cells. Then PHA-stimulated T cells or PBMCs were cultured during 4 to 8 days in complete medium alone or in the presence of anti- CD3/CD28-coated beads (Invitrogen). In co-culture proliferation experiment, PHA-stimulated T cells or PBMCs, plus or minus anti-CD27 antibody (clone LG.3A10 from eBioscience or 0323 from BioLegend) (K^g.ml^-1) from or ibrutinib (5 to 500nM) (Selleckchem), were co- cultured in the presence of 45 Gy irradiated autologous or allogenous LCLs that have been previously incubated with ^g ml^"1 of anti-CD3 antibody (OKT3) or not for one hour. Cells were co-cultured at ratio of one T cells or PBMC for 10 LCLs (ratio 1/10). Cell-proliferation was monitored by labeling T cells or PBMCs with the CellTrace violet dye (Violet Proliferation Dye 450, BD Biosciences) prior to stimulation or co-culture with LCLs, according to the manufacturer's instructions. After 8 days of culture, cells were harvested and CellTrace violet dye dilution was assessed by flow cytometry.

Flow cytometry. Cell staining and the flow cytometry based phenotypic analyses of PBMCs, blast T cells and cell lines were performed according to standard flow cytometry methods. The following mAbs with their identification numbers in brackets were conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), phycoerythrin-cyanin5 (PE-Cy5), phycoerythrin-cyanin5.5 (PE-Cy5.5), phycoerythrin-cyanin7 (PE-Cy7), Peridinin-chlorophyll (PerCP), Peridinin-chlorophyll-cyanin5.5 (PerCP-Cy5.5), allophycocyanin (APC), allophycocyanin-Cyanin7 (APC-Cy7), allophycocyanin- Vio7 (APC-Vio7), alexa-700, Brilliant Violet 421 (BV421), Brilliant Violet 510 (BV510), Brilliant Violet 650 (BV650), Brilliant Violet 785 (BV785): anti-CD3 (UCHT1), anti-CD4 (OKT4), anti-CD8 (RPA-T8), anti-CD 14 (M5E2), anti-CD 19 (HIB19), anti-CD25 (BC96), anti-CD27 (LG.3A10, M-T271, 0323), anti- CD28 (CD28.2), anti-CD31 (WM59), anti-CD56 (HCD56), anti-CD70 (113-16), anti- CD107a/b (H4A3/HAB4), anti-CD161 (HP-3G10), anti-CD197 (G043H7), anti-TCR V 7.2 (3C10), all purchased from Sony Biotechnology Inc. , anti-CD 16 (3G8), anti-CD45RA (HI 100), anti-CD45PvO (UCHL1), anti-CD95 (DX2), anti-TCRalphabeta, IgD (IA6-2), IgM (G20-127) from BD biosciences and anti-CD21 (BL13), anti-TCRgammadelta), anti-TCR Valpha24 (CI 5), anti-TCR Vbeta from Beckman coulter. Binding of CD27 to CD70 expressing-cells was assessed by incubation with Human CD27-muIg/Biotin Fusion Protein (Ancell Corporation) according to manufacturer's protocol and standard flow cytometry methods. Binding of CD27-muIg to cells was detected with PE-conjugated streptavidin (BD biosciences). All data were collected on FACS-Canto II or LSR-Fortessa cytometers (both from BD Biosciences) and analyzed using Flowjo Version 10.0.8rl software (Tree Star).

Calcium flux analysis. Ca²⁺ responses were assessed by flow cytometry, as previously described⁸. Briefly, cells were loaded with 5 μΜ Indo-1 AM (Molecular Probes) in presence of IX PowerLoad™ (ThermoFischerScientific) and 50nM of ibrutinib or not, washed, incubated with anti-CD4-APC and anti-CD8-PE mAbs, stimulated in the presence or not of 50nM of Ibrutinib by 5 μg ml ¹ of anti-CD3 (OKT3), anti-CD27 (clones LG.3A10, LG.7F9 (eBioscience) or 0323) or anti-CD28 (CD28.2) antibodies cross-linking with 10 μg ml^"1 of F(ab')₂ rabbit- anti-mouse IgG only adsorbed on human (Jackson Immunoresearch) and then incubated with ionomycin (5 μΜ). Cells were analyzed with a FACS ARIA flow cytometer (BD Biosciences). Changes in the intracellular calcium concentration are quantified by a shift in the indo-1 emission peak from 485 nm (indo-blue) for unbound dye to 405 nm (indo-violet) when the indo-1 molecule is bound to calcium. Ca²⁺ flux data were obtained using kinetic analyses of FlowJo software package (TreeStar). Intracellular Ca²⁺ levels correspond to the normalized ratio of 405 nm/485 nm indo-1 emission peaks.

Cytokine production and detection. For intracellular staining of cytokines, cells were re-stimulated overnight with irradiated LCLs beforehand preincubated with ^g ml^"1 of OKT3 and in the presence brefeldin A (GolgiPlug, BD). Cells are then fixed and permeabilized using the BD cytofix/cytoperm plus kit (BD Pharmingen) according to manufacturer's instructions. Next, cells are labeled with PE/Cy7-anti-TNF-a (mouse IgGl; MAbl l), APC-anti-IFN-γ (mouse IgGl, 4S.B3) and isotype-matched monoclonal antibodies purchased from Bio-Legend and analyzed by flow cytometry. Thereafter, cells were collected, washed and stained with BV785-anti-CD3, BV650-PE-anti-CD8 and BV510-anti-CD4 mAbs and analyzed by flow cytometry.

EBV-specific T cells detection. EBV-specific CD8⁺ T cells from PBMCs were co- cultured with 45 Gy irradiated LCLs for 8-10 days and were detected using a mix of unlabelled EBV HLA-A2:01 Pro5® Pentamers (Proimmune) mixed with R-PE Pro5® Fluorotag in addition with BV785-anti-CD3, BV650-PE-anti-CD8 and BV510-anti-CD4 monoclonal antibodies. The EBV HLA-A2:01 Pro5® Pentamers mix contains 3 different pentamers presenting FLYALLALLL (residues 356-364 from LMP2), CLGGLLTMV (residues 426-434 from LMP2) or GLCTLVAML (residues 259-267 from BMLF-1) peptides derived from LMP2 and BMLF1 proteins of EBV. Following their isolation, EBV-specific T cells activated phenotype is characterized using PE/Cy7-anti-CD25 and FITC-CD45RA antibodies. All staining are done according to manufacturer's instructions.

Cell transfection and CRISPR Cas9 genome editing. Full length cDNA encoding wild-type CD70 and CD70 R179X were obtained by RT-PCR from blasts of control donors and the patient respectively. The cDNAs were verified by sequencing and inserted into bicistronic lentiviral expression vector encoding the green fluorescent protein (GFP) as a reporter (pLenti7.3/V5-TOPO, Invitrogen) or a N-terminal Flag-tagged expression vector (p3XFLAG- myc-CMV-26, Sigma- Aldrich). Patient's LCLs were transduced with the pLenti7.3/V5-TOPO containing wild-type CD70 or the empty vector as previously described⁴. The lentiCRISPR plasmid was a gift from Feng Zhang (Addgene plasmid # 49535). All sgRNAs were designed using M IT CRISPR Design Tool (http ://crispr .mit. edu) . Three pairs of 24-bp forward (F) and reverse (R) of oligonucleotides targeting different sequences in the exon 3 of CD70 were synthesized (Eurogentec) with a 4-bp overhang to enable cloning into the Bsmbl site in reverse oligonucleotides and a 4-bp overhang containing the PAM sequence in forward oligonucleotides. sgRNAs sequences. Pairs of synthetized oligos was annealed, phosphorylated, ligated to linearized vector and transformed into Stbl3 bacteria (Life Technologies). sgRNAs insertion was confirmed by Sanger sequencing using sequencing primer. The lentiCRISPR plasmids were transduced by infection in LCLs and transfected by electroporation with Nepa21 electroporator (Nepagene) in Raji cells. LCLs CD70-positive and CD70-negative populations were enriched by sorting.

Immunoblotting and pull-down assays. Cells (5xl0⁶ cells/ml) were stimulated by anti-CD27 (monoclonal antibody LG.3A10) or anti-CD3 (monoclonal antibody OKT3) antibody (1 μg ml^"1) cross-linking with a rabbit-anti-mouse IgG (2μg ml^"1) for the indicated time periods. Cells were then lysed in 1% NP40, 50 mM Tris pH 8, 150 mM NaCl, 20 mM EDTA, 1 mM Na3V04, 1 mM NaF and complete protease inhibitor cocktail (Roche), as previously described⁸. Protein concentrations were quantitated by BCA assay (BIO-RAD). 80 μg of proteins were separated by SDS-PAGE and transferred on PVDF membranes (Millipore). For testing expressiMembranes were blocked with milk or BSA for 1 h before incubation with primary antibodies for 90 minutes. The following mAbs and rabbit polyclonal antibodies were used for immunoblotting: anti-ITK (clone 2F12), anti-phosphorylated PLC-γΙ (clone D6M9S), anti-phosphorylated ER l/2 (clone D13.14.4E) and anti-phosphorylated tyrosine (clone PY- 100) purchased from Cell signaling and, anti-FLAG (clone M2) and rabbit polyclonal anti- ACTIN antibody was from Sigma. Membranes were then washed and incubated with anti- mouse or anti-rabbit HRP-conjugated secondary antibodies from Cell Signaling and GE Healthcare, respectively. Pierce ECL western blotting substrate was used for detection. Binding assays using GST proteins were performed as outlined previously⁵⁰, using lysates from unstimulated cells, anti-CD3 or pervanadate stimulated cells. GST-fusion proteins with the SH3 domains of human FYN, ITK and LCK were obtained by RT-PCR and subcloning into pGEX- 2T vector (GE Healthcare).

Results:

Clinical and immunological features

We investigated an 8 year-old male of Egyptian origin with chronic EBV infection and lymphoma. His parents were first cousins. At the age of 3 years and 8 months, he was diagnosed with EBV-positive nodular sclerosing Hodgkin's lymphoma. He received chemotherapy and radiotherapy and achieved a complete response with no relapse since then. Since the age of 4.5 years, he had recurrent fever, lymphadenopathies and hepatosplenomegaly, which were associated with a high load of EBV in blood and polyclonal expansion of B cells in lymph nodes and spleen. His symptoms were relieved after the initiation of anti-CD20 therapy, leading to a decrease in EBV viral load, but reappeared requiring repeated anti-CD20 infusions and finally allogeneic hematopoietic stem cell transplantation. No other viral and bacterial infections were noted.

Immunological investigations have been carried out at the age of 5 years prior to anti- CD20 therapy and revealed significant but minor abnormalities including decreased counts of NK, iNKT, MAIT and memory B cells and reduced serum IgM and IgA levels. T-cell proliferation to CD3, PHA and antigen stimulations were normal. Based on these observations, it was suspected that the high susceptibility to EBV infection associated with early onset Hodgkin lymphoma was the consequence of an inborn error leading to immunodeficiency in the patient.

Identification of homozygous nonsense mutation in CD70

To identify the genetic basis of the immunodeficiency in the patient, we first tested known autosomal genetic defects causing susceptibility to EBV infection. No mutation in ITK, CD27, MAGT1 and CTPS1 was detected, excluding their causative role. We next performed whole-exome sequencing (WES). Because of the consanguinity of the parents, we focused our analysis on homozygous genetic variations. Only one homozygous variation was found predicted to be deleterious and localized in the CD70 gene. This variation consisting of a nonsense mutation (p.R179X, c.535C>T) in the third exon was only present as a heterozygous mutation in public database of exomes and in our institute database with a frequency of 1.48x 10^" ⁵ (see Methods). Sequencing by the Sanger method of CD70 in the kindred confirmed that the patient was homozygous for the c.535C>T mutation, while his parents and two healthy sisters were heterozygous carriers. At the protein level, the p.R179X mutation removes the last 15 C- terminal amino acids of CD70. 3D structure modeling of the human CD70 protein using as templates the TNF-like receptor Apo2L/TRAIL 3D structures (pdb 1DG6 and pdb 1D0G) showed that the last internal β-strand H was lost in the truncated CD70^R179X protein. Of note, in Apo2L, this strand is involved in the contacts formed between the different subunits of the homotrimer²⁸. Importantly, CD70 is known to be the ligand for CD27 molecule. Deficiency in CD27 causes a high susceptibility to EBV infection and the associated lymphoproliferative disorders⁵' ⁶' ²⁰. Thus, we considered CD70 as a strong candidate gene underlying the immunodeficiency of the patient.

Loss-of-expression and lack of binding to CD27 of the CD70 mutant protein

The c.535C>T mutation had no impact on the amount of CD70 mR A detected by qPCR in the PHA-stimulated T cells and in EBV-transformed B cells from the patient (further designated as lymphoblastoid cell line (LCL)) (data not shown). At the protein level, anti-CD70 antibody failed to detect CD70 by flow cytometry at cell surface of PHA-stimulated T cells of the patient. In contrast, expression of CD70 was detectable on a fraction of PHA-stimulated T cells from healthy donors at day 8. This proportion of CD70⁺ T cells increased in culture with a large proportion of T cells expressing CD70 at day 15. Similarly, CD70 was not detected on LCLs derived from the patient, in contrast to LCLs from healthy donors that expressed high levels of CD70 on their surface. Defective expression of CD70 was confirmed by analyzing the capacity of CD70^R179X to bind to CD27. A fusion protein containing the extracellular domain of CD27 (Fc-CD27) failed to bind on the surface of PHA-T cell blasts and LCLs from the patient, whereas its binding on control cells was detected. Similar results were obtained when wild-type CD70 or CD70^R179X proteins were transiently expressed in HEK-293 T cells. Expression of CD70 and CD70^R179X in HEK-293 T cells was further examined by western blot using N-terminus FLAG-tagged CD70 forms. The mutant CD70^R179X was weakly expressed compared to wild type CD70. Taken together, these results indicate that the p.R179X mutation in CD70 compromises its expression and has a deleterious effect on its ability to recognize its cognate ligand CD27. Therefore, we conclude that the CD70 deficiency mimics that of CD27 deficiency and thus likely accounts for the high susceptibility to EBV infection of the patient.

High levels of CD70 on activated B cells and EBV-infected B cells

In order to better understand the role of CD70 in anti-EBV immunity, we first analyzed the expression of CD70 in peripheral blood mononuclear cells (PBMCs) of several healthy donors. In humans, CD70 expression has been shown to be restricted to some DCs and B cell subsets, while CD27 is expressed on most T cells and memory B cells²³' ²⁹. Indeed, expression of CD70 was barely detectable on CD4 and CD8 T cells, monocytes, DC cells and neutrophils, with the noticeable exception of a small fraction of B cells. This contrasts with the high levels of CD27 on the surface of CD4⁺ and CD8⁺ T lymphocytes and a small fraction of B cells. CD70 expression on B cells was rapidly upregulated upon activation by a combination of phorbol 12- myristate 13-acetate (PMA) and ionomycin (Iono). At day 3 of stimulation, more than 80% of B cells expressed large amounts of CD70 in contrast to T cells. Similarly to activated B cells, all EBV-transformed B cell lines that we tested expressed high levels of CD70. These data suggest that the expression of CD70 is inducible in B cells in the course of EBV infection. To test this possibility, we analyzed the expression of CD70 in tonsils from two individuals with infectious mononucleosis. CD70 staining was found on large cells that were positive for Epstein-Barr Encoded RNA (EBER) and PAX5 specific markers of EBV and B cells respectively and corresponded to EBV -infected B cells. In contrast, CD27 expression was not associated with PAX5 and EBER staining and accumulated in cells located in the T-cell areas. These data indicate that expression of CD70 is upregulated in activated and in EBV-infected B cells, while most T cells express high amounts of CD27 at resting state. Therefore, CD27-CD70 interactions can occur between CD27-expressing T cells and CD70-expressing B cells during the course of an EBV infection and may play an important role in the control of EBV-activated B cells by T cells.

Decreased cytolytic response of patient T-cells against autologous EBV- transformed B cells

In mice, CD70 and CD27 play an important role in antigen specific-T cell responses, in particular during anti-viral responses²⁶' ³⁰. Hence, T-cell responses against EBV might be impaired in the absence of CD70. To test this hypothesis, we analyzed T-cell responses against EBV from PBMCs of the patient or healthy controls that were co-cultured with irradiated autologous LCLs. Proportions of CD4⁺, CD8⁺ as well as expression of activation markers were comparable when analyzed after four to five weeks of co-culture. After four weeks of co- culture, cell-cytotoxicity response and IFN-γ production by T cells was assessed against autologous EBV-transformed B cell lines (Fig. la). Compared to control T-cell cultures, T cells of the patient exhibited a markedly decreased cytotoxic activity and IFN-γ production, indicative of impaired T-cell responses to EBV. To formally prove the link between the CD70 deficiency on EBV-transformed B cells and the defective T-cell responses against EBV- transformed B cells, we carried out reconstitution experiments by transducing LCLs of the patient with a lentiviral vector containing a cDNA coding wild-type CD70 (or an empty vector), which induced surface expression of CD70 to levels comparable to those seen on LCLs from control donors (Fig. lb). In these conditions, the cytolytic response of patient's T cells towards autologous CD70-expressing LCLs was restored to a magnitude similar to that of control T cells (Fig. lc). Taken together, these results demonstrate that the lack of CD70 on patient's B cells results in impaired T-cell cytolytic responses towards EBV-infected B cells. However, the intrinsic cytotoxicity function of patient's T cells was preserved, while ectopic CD70 expression on target cells did not increase killing by T cells.

Impaired proliferation of patient EBV-specific T cells We next tested the possibility that impaired T-cell cytotoxicity and cytokine production towards autologous CD70-deficient LCLs in the patient was the consequence of a defect in EBV-specific T-cell expansion. HLA genotyping of the patient showed that he was carrier of HLA-A*01, for which no tetramer reagents were available to directly assess EBV-specific T cells. However, EBV-induced proliferating T-cells were detectable by cell trace violet dye dilution and expression of the activation marker CD25 among PBMCs of patient, when co cultured with autologous LCLs in which CD70 expression was restored, but not with CD70- deficient parental LCLs (Fig. Id, upper panels). Proliferating T cells from a HLA-A*01- expressing healthy donor were also observed when co-cultured with HLA-A*01 CD70- expressing LCLs of the patient, but not with HLA-A*01 CD70-deficient LCLs (middle up panels). In contrast, in non autologous conditions, only few proliferating T-cells were detected among PBMCs from healthy donor carriers of HLA-A*02 and HLA-A*03 alleles (middle low and lower panels).

Defective T-cell expansion of EBV-specific T cells in the absence of CD70 on B cells. To next prove the role of CD70 in anti-EBV immunity, CD70-deficient LCLs were derived from PBMCs of a healthy HLA- A* 02 individual by gene inactivation. Most HLA- A* 02 individuals develop EBV-specific T cells against the GLCTLVAMV peptide (also termed GLC epitope), which is derived from the EBV-lytic cycle protein BMLF1³¹. CD70-deficient LCLs were obtained by blunting CD70 expression using CRISPR (clustered regularly interspaced short palindrome repeats)-associated nuclease Cas9 technology. Three different CRISPR-Cas9 constructs containing RNA guides targeting exon 3 of CD70 were transiently transfected in the LCLs. After having been selected and sorted to enrich for CD70-negative cells, three stable CD70-deficient (CD70KO) LCLs cell lines were obtained (hereafter referred as CD70KO- CRISPR1, CD70KO-CRISPR2 and CD70KO-CRISPR3). These cell lines expressed comparable amounts of HLA- A* 02 molecules on their surface when compared to those of the wild-type parental LCLs or LCLs transfected with an empty CRISPR-Cas9 construct. We then examined the expansion of EBV-specific T cells of the HLA-A*02 healthy donor in coculture experiments with autologous irradiated wild-type LCLs (transfected with an empty CRISPR- Cas9 construct) or CD70KO-CRISPR1 LCLs. EBV-specific T cells were detectable at day 0 using a mix of pentamers for GLC, CLG and FLY epitopes, and following co culture with wild- type CD70-expressing LCLs (Empty CRISPR LCLs), their proportion increased at day 8 of culture by 5-fold. Most of these cells expressed CD25 and were strongly proliferative (data not shown). In striking contrast, culture of T cells with autologous CD70KO-CRISPR1 LCLs did not result in any expansion of EBV-specific T cells. Similar results were obtained with PBMCs from two other healthy HLA-A*02 donors (Ctr.2 and Ctr.3). No such expansion was observed with T cells from an EBV naive donor (Ctr.4). Absence of expansion was also observed when HLA-A* 02 PBMCs were co cultured with both other CD70-deficient LCLs CD70KO- CRISPR2 and CD70KO-CRISPR3. Our observations thus demonstrate that CD70 is a key factor required for the expansion of EBV-specific T cells. Furthermore, control and patient T cells expanded in the presence of autologous CD70-expressing LCLs, were able to kill efficiently both CD70-expressing and CD70-deficient LCLs, indicating that CD70 deficiency per se does not preclude cytotoxic activity of T cells (Figure 2). In contrast, T cells that were co cultured with autologous CD70-deficient LCLs had no killing activity demonstrating that specific effector T-cells require CD70 for expansion.

CD70 on B cells provides a costimulatory signal required for TCR-mediated proliferation

To further extend our observation and to formally prove that CD70 deficiency results in blunted proliferation and activation in response to TCR stimulation by B-LCLs, we analyzed T-cell proliferative responses after stimulation with irradiated LCLs expressing or not CD70 in the presence of anti-CD3 antibody. Importantly, in these conditions activation of T cells was not restricted to HLA-A molecules expressed by irradiated LCLs. At day 8, only a low proportion of T cells from the patient or a healthy donor were detectable in the coculture with CD70-deficient LCLs obtained by CRISPR (CD70-CRISPR1 LCLs) or CD70-deficient LCLs from the patient. In contrast, when PBMCs were co cultured with LCLs from the patient in which the expression of CD70 was restored or CD70-expressing LCLs from a healthy donor (Empty CRISPR LCLs), most of T cells from both patient and the healthy donor were able to divide. A large proportion of these proliferating T cells expressed high levels of CD27 and CD25. Dividing T cells included both CD4⁺ and CD8⁺ T cells, although the proportion of CD8⁺ cells was higher. These proliferating T cells displayed an effector phenotype since a majority of them had the capacity to secrete IFN-gammaalpha upon re-stimulation. The results were reproduced and extended with T cells from different healthy donors harboring different HLA haplotypes including HLA-A1, HLA-A2 and HLA- A3. In these conditions, proliferation of T cells was dependent of CD3 stimulation, and in most individuals, a vast majority of T cells had achieved at least one division when co cultured with CD70-expressing LCLs but not with CD70-deficient LCLs. In addition, this proliferation was substantially reduced by addition of anti-CD27 antibody as expected. These results demonstrate that CD70 provides an essential costimulatory signal to TCR-mediated proliferation of T cells when expressed on B cells.

CD27-mediated T cell proliferation is dependent of ITK Because CD27, ITK, and CD70 deficiencies appear as phenocopies, we hypothesized that CD27-CD70 and ITK could form a functional and molecular cluster. To test it, we first examine CD70-dependent T-cell proliferation of PBMCs from CD27-deficient and ITK- deficient patients: four newly described patients, including one patient carrier of a homozygous mutation p.Wl 10X in CD27, two patients with a homozygous mutation p.R29C in ITK (Pat.l ITK^" and Pat.2 ITK^") and one patient with a homozygous mutation p.L38P in ITK (Pat.3 ITK^"). Assessing the possible impact of these mutations on a model of the ITK 3D structure, made on the basis of the BTK 3D structure (pdb: lB55), indicated that they may impair the capacity of the PH domain to interact with phosphoinositols similarly to the R29H mutation as reported previously¹⁷.

The Wl 10X mutation abolished the surface expression of CD27 on PBMCs and T-cell blasts of the patient. As expected, proliferation of CD27-deficient T-cell blasts in response to irradiated CD70-expressing B-LCLs in the presence of anti-CD3 antibody was markedly reduced. Expression of ITK was strongly diminished in lysates from T cells of patients carrying R29C and the L38P mutation. When examined, T-cell proliferation of ITK-deficient PBMCs or T-cell blasts in response to irradiated CD70-expressing LCLs in the presence of anti-CD3 antibody was markedly reduced. Importantly, ITK-deficient T cells expressed CD27 levels comparable to those of control T cells or CD70-deficient T cells and displayed normal proliferation in response to CD3 or CD3 plus CD28 stimulations. Collectively, these data indicate that the costimulatory signal triggered by CD27 on T cells when engaged by CD70 on B cells is dependent of the tyrosine kinase ITK. In accordance, addition of Ibrutinib an inhibitor of TEC kinases³² including ITK inhibited CD70-expressing LCL-mediated T-cell proliferation.

To further decipher the role of ITK in CD27 signaling, we next analyzed the activation events that follow the CD27 engagement. Stimulation of human T cells with anti-CD27 antibody or soluble CD70 has been reported to result in calcium mobilization in na^'ive T cells and γδ T cells³³' ³⁴. In mice, ITK is also known to activate PLC-γΙ and the subsequent calcium flux following TCR activation¹⁶' ¹⁸. Calcium mobilization in T cells in response to stimulation by anti-CD3 antibody or anti-CD27 antibody was thus investigated. ITK-deficient PBMC derived T cells and T-cell blasts exhibited reduced calcium mobilization upon CD3 stimulation, in contrast to cells from healthy donors, CD70-deficient or CD27-deficient patients. Stimulation of CD27 also induced calcium mobilization in T cells and T-cell blasts from the CD70-deficient patient and healthy donors. The magnitude of the calcium signal triggered by CD27 was often less important and consistently delayed compared to the signal triggered by CD3 stimulation. In these conditions, anti-CD28 stimulation did not induce any calcium flux. In contrast, CD27 stimulation of ITK-deficient or CD27-deficient T cells resulted in a very weak calcium mobilization. Likewise, Ibrutinib completely inhibited CD27-mediated calcium mobilization in T cells and T-cell blasts from healthy donors, while CD3-mediated calcium mobilization was partially blocked to a magnitude equivalent to that observed in ITK-deficient cells. These results demonstrate that ITK is required for CD27-mediated calcium mobilization.

The precise mechanism involved in CD27 signaling has not been yet clearly defined although a role for TRAF molecules has been reported³⁵. Interestingly, CD27 contained in its intracellular domain several proline and tyrosine residues, which may form potential docking sites for the SH3 and SH2 domains of ITK, respectively. Notably, the NH2 -terminal region has pro line-containing sequences sharing similarities with the consensus sequence involved in the interaction with the SH3 domain of ITK³⁶. These sequences in CD27 are highly conserved in mammals. We thus examined whether ITK could functionally or directly interact with CD27. We first tested the capacity of CD27 to trigger tyrosine phosphorylation events. Tyrosine phosphorylated proteins were detected in lysates from control T-cell blasts of a healthy donor, 15 minutes after stimulation by anti-CD27 antibody. These phosphorylation signals were in part distinct and delayed compared to the signals observed in response to CD3 stimulation. Interestingly, a protein with the size of ITK was phosphorylated upon CD27 but not CD3 stimulation. Further analysis of signals known to be dependent of ITK¹⁶, such as phosphorylation of PLC-γΙ and ERK1/2 showed that both pathways were activated upon CD27 stimulation, albeit less intensively than in response to CD3 stimulation. Evidence of a direct association between ITK and CD27 was then searched for by pull-down assays with GST fusion proteins containing the SH3 domain of ITK, FYN or LCK that are the most important SH3- containing tyrosine kinases involved in TCR activation. CD27 was precipitated from PBMCs and T-cell blast lysates with the GST containing the SH3 domain of ITK. In contrast, no association or only a moderate association was observed with the GST alone or the GST containing the SH3 domain of FYN or LCK. These data strongly suggest that ITK has the capacity to directly interact with CD27 via its SH3 domain, coupling CD27 to downstream signaling pathways.

Discussion:

In this study, we report for the first time a genetic deficiency in CD70 in a patient with defective immunity to EBV infection associated with uncontrolled lymphoproliferation of infected B-cells. We decipher the pathway involving CD70 and its ligand CD27 and provide the evidence that CD70, CD27 and ITK molecules form a functional cluster that plays a crucial role in the immune surveillance of B cells by T cells. When expressed on B cells, CD70 delivers signals to T cells through CD27 and ITK, those being necessary for T-cell proliferation maintenance and hence execution of the effector programs.

The patient had recurrent B-cell lymphoproliferation associated with a high viral load of EBV and developed EBV-associated Hodgkin's lymphoma. His susceptibility to infections appears to be restricted to EBV as until today he has not suffered from other viral or bacterial infections. Based on a single patient who in addition is still young, it is obviously difficult to firmly conclude on the clinical consequences associated with CD70 deficiency. However, genetic defects in CD27, which is the only known ligand for CD70, have been reported in 17 patients⁵' ⁶' ²⁰. Notably, all of them developed EBV-related disorders as initial and main clinical manifestations, including lymphoproliferation disorders like Hodgkin's lymphoma and diffuse large B-cell lymphoma. Therefore, the clinical symptoms of the CD70-deficient patient are similar if not identical to those associated with the CD27 deficiency. Hence, CD70 deficiency appears to be a phenocopy of the CD27 deficiency. Phenotypes of CD27- and CD70-deficient mice are also comparable, and both characterized by diminished antiviral responses²²' ²⁵' ²¹. Interestingly enough, the clinical phenotype associated with ITK-deficiency is also very close to the one of CD27 and CD70 deficiencies⁷.

Our results indicate that the CD70-CD27 axis represents a key factor of the immune response to EBV in humans. We showed that CD70 on EBV -infected B cells provides crucial signals to EBV-specific CTLs for their expansion. We observed that CD70 is strongly upregulated in activated B cells and EBV-infected B cells. CD70 is also constitutively expressed by many EBV- and non EBV-dependent B cell malignancies²⁴. The peculiar predisposition to EBV infection associated with CD27-, CD70- and ITK-deficiencies likely results from the unique tropism of EBV to B cells and its capacity to induce B-cell proliferation and transformation. An efficient in vivo immune response to EBV requires rapid and massive expansion of effector cells to provide a matching amount of CTLs to eliminate highly proliferating EBV-infected B cells. The CD70-CD27-ITK axis is thus a critical component of this response.

Numerous studies in mice have documented the role of the CD27-CD70 axis in the generation and the maintenance of CD8⁺ T cell responses, including establishment of long-term memory²². The recent description of CD70-deficient mice confirmed the particular importance of CD70 in the initiation of primary CD8⁺ T-cell responses²⁶. Accordingly, acquisition of T- cell immunity towards EBV-infected B cells appears to be defective in the CD70-deficient patient, correlating with the absence of a proper expansion and proliferation of EBV-specific effector CD8⁺ T cells in vitro. However, memory T-cell immunity to EBV is likely preserved in the absence of CD70 since specific anti-EBV T cells could be expanded from PBMCs of the patient when co cultured with autologous CD70-expressing LCLs. Collectively, these results pinpoint to a specific role of CD70-CD27 interactions in triggering cell division of activated T cells without affecting their intrinsic capacity to execute their cytotoxic and IFN-y-producing functions and to differentiate into memory cells.

In accordance with previous studies, our observations demonstrate that CD27 behaves as a co stimulatory molecule of TCR-dependent lymphocyte activation²⁹. The co-signals delivered by CD27 are crucial for sustained T-cell expansion as recently highlighted³⁷. In addition, we showed that proliferation associated cosignals downstream of CD27 are strictly dependent on ITK that can bind to the intracytoplasmic tail of CD27 via its SH3 domain. It is well established that ITK is directly involved in TCR signaling, by its ability to activate PLC- γΐ, Ca²⁺ flux and ERK kinases¹⁶' ¹⁸. In accordance, CD27 engagement also triggers activation of PLC-γΙ, Ca²⁺ flux and ERK kinases, suggesting a quantitative amplification of the TCR strength by CD27 as suggested²⁹' ³⁷. More studies are warranted to decipher the exact molecular requirements for ITK in CD27 signaling and how it cooperates with TCR signaling to drive cell division.

Although our observations establish that CD70 on B cells is a key player in T-cell immunity towards proliferating B cells, CD70 is also expressed on other hematopoietic cells including sub populations of activated T cells and DC cells. In the different models previously tested, CD27-dependent priming and maintenance of T-cell responses was shown to involve interactions with CD70 expressed on DCs³⁸' ³⁹. Thus, it is also conceivable that the absence of CD70 on DCs contributes to the lack of control of EBV infection in the patient, in particular during the priming phase. Because CD27- and ITK-deficient patients did not develop major susceptibility to other infections, CD70 on DCs seems not to be absolutely required for immune responses to other pathogens. Partial redundancy with other TNFR family members such as 4- 1BB and OX40 that are expressed by T cells could provide similar signals to T cells as those deliver by CD27, when engaged by their ligand during interactions with DC²⁹' ⁴⁰.

Interestingly enough, the mechanism underlying the defective immunity to EBV in the SAP deficiency caused by mutations in SH2D1A (or XLP-1 syndrome) is distinct and is associated with a different clinical phenotype than the one associated with CD27- CD70- ITK- deficiencies. In SAP deficiency, impaired T-B cell interactions through SLAM receptors play a pivotal role in the susceptibility to EBV infection, resulting in hemophagocytic syndrome (or HLH) associated with an expansion of CD8⁺ T cells in the majority of patients³. Our results indicate that the CD27-CD70-ITK axis also plays an important role during T-B cell interactions in the course of EBV infection. However, the impaired control of EBV infection in patients with CD27- CD70- ITK-deficiencies did not lead readily to HLH. In the absence of SAP, effector-memory EBV-specific CD8⁺ T cells have been shown to exhibit decreased capacity to kill EBV -infected B cells¹¹' ¹², whereas cell expansion is normal or even increased because of defective activation-induced cell death¹³, in sharp contrast to CD27- CD70- ITK-deficiencies. Pathogenesis of XLP is thought to result from immunopathology caused by CD8⁺ T cells unable to kill B cell targets. This is consistent with the pathophysiology of HLH, which is known to depend of the accumulation of activated T cells producing large amounts of IFN-γ as the result of their inability to kill infected cells⁴¹. On the contrary, in CD27- CD70- ITK-deficiencies accumulation of activated T cells in the course of EBV infection is deficient, resulting from their impaired capacity to proliferate. These distinct molecular defects pinpoint to two critical steps in the induction of T cell response to EBV, i.e. cell expansion and cytotoxic effector function that depend on different molecules/pathways CD70/CD27/ITK and SLAM/SAP respectively for their execution.

Collectively, our observations suggest that CD70 on B cells act as a semaphore to signal abnormal B cell proliferation to T cells even in the absence of EBV as a trigger of B cell proliferation. This fits well with the observation that somatic mutations in CD70, including large deletions have been identified in B-cell lymphomas such as diffuse large B cell lymphomas and Burkitt's lymphomas⁴²' ⁴³' ⁴⁴. Notably, a recent report identified CD70 as one of the most frequently mutated genes in a series of diffuse large B-cell lymphomas⁴⁵. Accumulation of mutations in CD70 may represent a mechanism for malignant B cells to escape immune surveillance by T cells. Our data and previous studies in mice indicate that ectopic expression of CD70 on non-lymphoid tumor cells or CD70 expression in B cells (our report) can evoke T-cell mediated immunity against tumor cells irrespectively of their expression of CD70⁴⁶' ⁴⁷' ⁴⁸. Thus, CD70-CD27 could represent an important target to induce anti-tumoral vaccination. Restoration or induction of CD70 expression in some lymphoma cells lacking CD70 might represent a therapeutic approach to induce a global and potent anti-tumoral immunity.

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Claims

CLAIMS:

1. A method of treating a B-cell malignancy in a subject in need thereof comprising i) providing a sample of malignant B cells obtained from the subject ii) isolating and culturing a population of malignant B cells from the sample of step i), iii) introducing in the population malignant B cells of step ii) a nucleic acid molecule encoding for a CD70 polypeptide and iv) administering to the subject a therapeutically effective amount of the population of malignant B cells of step iii).

2. The method of claim 1 wherein the B cell malignancy is selected from the group consisting of non-Hodgkin's lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma, primary effusion lymphoma, diffuse large B-cell lymphoma, splenic marginal zone lymphoma, MALT (mucosa-associated lymphoid tissue) lymphoma, hairy cell leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, B cell lymphomas (e.g. various forms of Hodgkin's disease, B cell non-Hodgkin's lymphoma (NHL) and related lymphomas (e.g. Waldenstrom's macroglobulinaemia (also called lymphoplasmacytic lymphoma or immunocytoma) or central nervous system lymphomas), leukemias (e.g. acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL; also termed B cell chronic lymphocytic leukemia BCLL), hairy cell leukemia and chronic myoblastic leukemia), myelomas (e.g. multiple myeloma), small lymphocytic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, extra-nodal marginal zone B cell lymphoma of mucosa-associated (MALT) lymphoid tissue, nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma/leukemia, grey zone lymphoma, B cell proliferations of uncertain malignant potential, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorder.

3. The method of claim 2 wherein the B-cell lymphoma is caused by Epstein-Barr virus (EBV).

4. The method of claim 2 wherein the B cell lymphoma is a diffuse large B-cell lymphoma associated with mutation in the gene encoding for CD70.

5. The method of claim 1 wherein the malignant B cells are transformed with a nucleic acid molecule encoding for a polypeptide comprising an amino acid sequence having at least 90% of identity with SEQ ID NO: 1.

6. The method of claim 5 wherein the introduction of the nucleic acid molecule is performed with a retro-viral vector.

7. A vaccine composition comprising a population of malignant B cells transformed with a nucleic acid molecule encoding for a CD70 polypeptide.

8. The vaccine composition of claim 7 wherein the malignant B cells are transformed with a nucleic acid molecule encoding for a polypeptide comprising an amino acid sequence having at least 90% of identity with SEQ ID NO: l

9. The vaccine composition of claim 7 for use in the treatment of a B-cell malignancy.

10. The vaccine composition for the use according to claim 7 wherein the wherein the B cell malignancy is selected from the group consisting of non-Hodgkin's lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma, primary effusion lymphoma, diffuse large B-cell lymphoma, splenic marginal zone lymphoma, MALT (mucosa- associated lymphoid tissue) lymphoma, hairy cell leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, B cell lymphomas (e.g. various forms of Hodgkin's disease, B cell non-Hodgkin's lymphoma (NHL) and related lymphomas (e.g. Waldenstrom's macroglobulinaemia (also called lymphoplasmacytic lymphoma or immunocytoma) or central nervous system lymphomas), leukemias (e.g. acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL; also termed B cell chronic lymphocytic leukemia BCLL), hairy cell leukemia and chronic myoblastic leukemia), myelomas (e.g. multiple myeloma), small lymphocytic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, extra-nodal marginal zone B cell lymphoma of mucosa-associated (MALT) lymphoid tissue, nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma/leukemia, grey zone lymphoma, B cell proliferations of uncertain malignant potential, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorder.