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  • C12N2501/23—Interleukins [IL]
  • C12N2501/2321—Interleukin-21 (IL-21)

Definitions

• the present invention in some embodiments thereof, relates to non-graft versus host disease (GVHD) inducing anti-third party cells comprising a central memory T- lymphocyte phenotype and, more particularly, but not exclusively, to the use of same for graft versus leukemia/lymphoma treatment.
• GVHD non-graft versus host disease
• donor CD8+ T cells present in the stem cell graft or in DLI have the added benefit of GVL effect that can kill residual malignant cells [Ho WY et al., J Clin Invest. (2002) 110: 1415-1417].
• this benefit is offset by graft- versus-host disease (GVHD), associated with CD8 T cells, which adversely impact transplant-related mortality.
• GVHD graft- versus-host disease
• activated anti-3rd party CD8+ cells with central memory phenotype can further support and improve bone marrow (BM) engraftment, likely due to the improved lymph node homing of the Tcm cells, their proliferative capacity and prolonged persistence in BMT recipients [Ophir E et al., Blood (2010) 115: 2095-2104].
• BM bone marrow
• the present inventors have shown that in humans, anti-3rd party CTLs, although depleted of alloreactivity, exhibit potent in-vitro killing of B-CLL and different types of primary lymphoma cells [Lask A et al.
• a method of treating a disease in a subject in need thereof comprising: (a) transplanting a non-syngeneic cell or tissue graft to the subject; and (b) administering to the subject a therapeutically effective amount of an isolated population of cells comprising non-graft versus host (GVHD) inducing anti-third party cells having a central memory T-lymphocyte (Tcm) phenotype, the cells being tolerance-inducing cells and capable of homing to the lymph nodes following transplantation, and further wherein the cells are either: (i) non-syngeneic with both the subject and the graft; or (ii) non-syngeneic with the graft and syngeneic with the subject, thereby treating the subject.
• GVHD non-graft versus host
• Tcm central memory T-lymphocyte
• an isolated population of cells comprising non-graft versus host (GVHD) inducing anti-third party cells having a central memory T-lymphocyte (Tcm) phenotype, the cells being tolerance-inducing cells and capable of homing to the lymph nodes following transplantation, as an adjuvant treatment for eradication of a disease in a subject who has been transplanted with a non-syngeneic cell or tissue graft, wherein the cells are either: (i) non-syngeneic with both the subject and the graft; or (ii) non- syngeneic with the graft and syngeneic with the subject.
• GVHD non-graft versus host
• Tcm central memory T-lymphocyte
• an isolated population of cells comprising non-GVHD inducing anti-third party cells having a central memory T-lymphocyte (Tcm) phenotype, the cells being tolerance-inducing cells and capable of homing to the lymph nodes following transplantation, and further wherein the cells are either: (i) non-syngeneic with both a host subject and a graft, wherein the subject and the graft are non-syngeneic; or (ii) non- syngeneic with a graft and syngeneic with a host subject, wherein the subject and the graft are non-syngeneic.
• Tcm central memory T-lymphocyte
• a method of treating a disease in a subject in need thereof comprising: (a) transplanting immature hematopoietic cells to the subject; and (b) administering to the subject a therapeutically effective amount of an isolated population of cells comprising non-graft versus host (GVHD) inducing anti-third party cells having a central memory T-lymphocyte (Tcm) phenotype, the cells being tolerance-inducing cells and capable of homing to the lymph nodes following transplantation, wherein when the immature hematopoietic cells are syngeneic with the subject, the isolated population of cells are selected syngeneic with the subject or non-syngeneic with the subject.
• GVHD non-graft versus host
• Tcm central memory T-lymphocyte
• a method of treating a disease in a subject in need thereof comprising: (a) transplanting immature hematopoietic cells to the subject; and (b) administering to the subject a therapeutically effective amount of an isolated population of cells comprising non-graft versus host (GVHD) inducing anti-third party cells having a central memory T-lymphocyte (Tcm) phenotype, the cells being tolerance-inducing cells and capable of homing to the lymph nodes following transplantation, wherein when the immature hematopoietic cells are non-syngeneic with the subject, the isolated population of cells are selected syngeneic with the subject or non-syngeneic with both the subject and the immature hematopoielic cells.
• the non-syngeneic cell or tissue graft is derived from a donor selected from the group consisting of an HLA identical donor and
• the subject and the donor are both humans.
• the subject is a human subject.
• the cell or tissue graft is non- autologous and the isolated population of cells are autologous.
• the cell or tissue graft and the isolated population of cells are from different donors.
• the disease comprises a malignancy.
• the malignancy comprises a B cell malignancy.
• the malignancy comprises a leukemia.
• the malignancy comprises a lymphoma.
• the graft comprises bone marrow cells.
• the bone marrow cells comprise immature hematopoietic cells.
• the immature hematopoietic cells are non-syngeneic with the subject, the isolated population of cells are syngeneic with the subject.
• the immature hematopoietic cells are non-autologous and the isolated population of cells are autologous.
• the isolated population of cells are non-syngeneic with both the subject and with the graft.
• the immature hematopoietic cells and the isolated population of cells are from different donors.
• the method further comprises administering at least one immunosuppressant drug to the subject.
• the subject is further conditioned under sublethal, lethal or supralethal conditioning prior to step (a).
• step (a) and step (b) are effected concomitantly.
• step (b) is effected prior to step (a).
• step (b) is effected one day after step (a).
• the central memory T- lymphocyte (Tcm) phenotype comprises a CD8 + /CD62L + signature.
• At least 50 % of the isolated population of cells have the signature.
• the lymph nodes comprise peripheral lymph nodes.
• the lymph nodes comprise mesenteric lymph nodes.
• the cells non-syngeneic with the graft and syngeneic with the subject comprise autologous cells.
• the cells non-syngeneic with the graft and syngeneic with the host subject are autologous.
• the immature hematopoietic cells and the isolated population of cells are autologous.
• the immature hematopoietic cells are autologous and the isolated population of cells are non-autologous.
• the immature hematopoietic cells are non-autologous and the isolated population of cells are autologous.
• the immature hematopoietic cells and the isolated population of cells are from different donors.
• the anti-third party cells having a central memory T-lymphocyte (Tcm) phenotype are generated by: (a) contacting peripheral blood mononuclear cells (PBMC) with a third party antigen or antigens in the presence or absence of IL-21 under conditions which allow elimination of GVH reactive cells; and (b) culturing the cells resulting from step (a) in the presence of IL-15 in an antigen free environment under conditions which allow proliferation of cells comprising the central memory T-lymphocyte (Tcm) phenotype, thereby generating the isolated population of cells.
• PBMC peripheral blood mononuclear cells
• Tcm central memory T-lymphocyte
• the conditions which allow elimination of GVH reactive cells comprise culturing for 1-5 days.
• the conditions which allow elimination of GVH reactive cells comprise 1-5 days in a culture deprived of cytokines.
• the third party antigen or antigens comprise dendritic cells.
• the third party antigen or antigens is selected from the group consisting of third party cells, a cell antigen, a viral antigen, a bacterial antigen, a protein extract, a purified protein and a synthetic peptide presented by autologous presenting cells, non-autologous presenting cells or on an artificial vehicle or artificial antigen presenting cell.
• the conditions which allow proliferation of cells further comprise IL-7.
• the culturing in the presence of IL-15 is effected for 3-30 days.
• the culturing in the presence of IL-15 is effected for 6-30 days.
• the culturing in the presence of IL-15 is effected for 3-20 days.
• the conditions which allow proliferation of cells comprise culturing for 3-10 days.
• the conditions which allow proliferation of cells comprise culturing for 3-7 days.
• FIGs. 1 A- 1 depict inhibition of tumor relapse by syngeneic derived anti-3 rd party Tcm cells after syngeneic bone marrow transplantation.
• BALB/c mice H-2 d
• BALB/c-NUDE H-2 d
• FIGs. 1 A- 1 depict inhibition of tumor relapse by syngeneic derived anti-3 rd party Tcm cells after syngeneic bone marrow transplantation.
• BALB/c mice H-2 d
• BALB/c-NUDE H-2 d
• FIGS. 1A-H are pictures depicting tumor growth monitored by bioluminescence imaging (BLI) from day 14 in weekly intervals;
• Figure II is a graph depicting the survival rate of the animals from the different treatment groups.
• X-axis indicates days after tumor cell injection and the y-axis indicates the proportion of recipient mice surviving.
• FIGs. 2A-G depict inhibition of tumor relapse by Fl derived anti-3 rd party CD8 T cells after syngeneic bone marrow transplantation.
• BALB/c mice H-2 d
• BALB/c-NUDE H-2 d
• FIGs. 2A-G depict inhibition of tumor relapse by Fl derived anti-3 rd party CD8 T cells after syngeneic bone marrow transplantation.
• BALB/c-NUDE H-2 d
• FIGs. 2A-G depict inhibition of tumor relapse by Fl derived anti-3 rd party CD8 T cells after syngeneic bone marrow transplantation.
• Figures 2A-F are pictures depicting tumor growth monitored by BLI from day 14 in weekly intervals.
• Figure 2G is a graph depicting the survival rate of the animals from the different treatment groups. X-axis indicates days after tumor cell injection and the y-axis indicates the proportion of recipient mice surviving.
• FIGs. 3A-I depict inhibition of tumor relapse by allogeneic derived anti-3 rd party
• mice received intravenously a transplant of 3 x 10 6 allogeneic T cell depleted bone marrow cells (C57BL/6-NUDE (H-2 b ) in the presence or absence of 5 x 10 3 A20-luc lymphoma cells (day 0). Mice were then intravenously injected with C57BL/6 derived cells (on day +1).
• Figures 3A-H are pictures depicting tumor growth monitored by BLI from day 13 in weekly intervals;
• Figure 31 is a graph depicting the survival rate of the animals from the different treatment groups.
• X-axis indicates days after tumor cell injection and the y-axis indicates the proportion of recipient mice surviving.
• FIGs. 4A-B are graphs depicting the proliferation and cell phenotype of anti-third party T central memory cells of the present invention.
• Figure 4A depicts Tcm cell proliferation from day 0 until day 12 of culture; and
• Figure 4B depicts cell phenotype using the same culture against Allo-DC.
• FIG. 5 is a graph depicting the percent of apoptotic cells after 22 hours of mixed lymphocyte reaction (MLR) with B-cell lymphoma and plasma cell leukemia cell lines.
• MLR mixed lymphocyte reaction
• CalceinAM pre-Iabeled Daudi, H.My2 CIR HLA A2 K66A mutant or L363 cell lines were incubated for 22 hours with or without 5-fold excess of anti-3 rd party Tcm.
• Annexin V+ cells were determined by FACS. Data is shown as meanlSD of pentaplicate cultures. ***p ⁇ 0.001 values indicate statistically significant changes compared to samples cultured in the absence of Tcm.
• FIG. 6 is a graph depicting the number of live cells after 22 hours of mixed lymphocyte reaction (MLR) with B-cell lymphoma EBV-LCL and plasma cell leukemia cell lines.
• MLR mixed lymphocyte reaction
• CalceinAM pre-labeled Daudi, H.My2 CIR HLA A2 K66A mutant or L363 cell lines were incubated for 22 hours with or without 5-fold excess of anti-3 rd party Tcm. Numbers of viable CalceinAM + cells were determined by FACS. Data is shown as mean ⁇ SD of pentaplicate cultures. ***p ⁇ 0.001 values indicate statistically significant changes compared to samples cultured in the absence of Tcm. DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
• the present invention in some embodiments thereof, relates to non-graft versus host disease (GVHD) inducing anti-third party cells comprising a central memory T- lymphocyte phenotype and, more particularly, but not exclusively, to the use of same for graft versus leukemia/lymphoma treatment.
• GVHD non-graft versus host disease
• anti-3rd party CD8+ T central memory (Tcm) cells comprise in vivo graft versus leukemia/lymphoma (GVL) activity and may therefore be used to treat malignant hematopoietic diseases, such as lymphoma and leukemia.
• Tcm T central memory
• the present inventors have shown GVL reactivity of anti-3rd party CD8+ Tcm cells in an in-vivo mouse model specifically designed to simulate allogeneic bone marrow transplant (BMT) in lymphoma patients.
• BMT bone marrow transplant
• the present inventors established a minimal residual disease mouse model for B cell lymphoma utilizing A20 B cell lymphoma cell line in which luciferase reporter gene was stably integrated into its genome (A20-luc). These cells enabled sensitive monitoring of in-vivo tumor progression by bioluminescence imaging (BLI). Using this model, inventors discovered that both syngeneic and allogeneic anti-3rd party Tcm cells exhibit marked GVL reactivity without causing graft versus host disease (GVHD) when administered in conjunction with a syngeneic bone marrow transplant (Examples 2 and 3, respectively, of the Examples section which follows).
• GVHD graft versus host disease
• the present inventors showed an effective GVL effect devoid of GVHD when administering allogeneic anti-3rd party Tcm cells (donor type) in conjunction with an allogeneic bone marrow transplant (Example 4, of the Examples section which follows).
• all these findings substantiate the use of anti-third party Tcm cells as graft versus leukemia/lymphoma cells for eradication of diseased cells.
• the present results demonstrate the ability to use anti-third party Tcm cells from a donor non-syngeneic with respect to the recipient and to the transplant donor (e.g. from two different donors).
• a method of treating a disease in a subject in need thereof comprising: (a) transplanting a cell or tissue graft to the subject; and (b) administering to the subject a therapeutically effective amount of an isolated population of cells comprising non-graft versus host (GVHD) inducing anti-third party cells having a central memory T- lymphocyte (Tcm) phenotype, the cells being tolerance-inducing cells and capable of homing to the lymph nodes following transplantation, and further wherein the cells are either: (i) non-syngeneic with both the subject and the graft; or (ii) non-syngeneic with the graft and syngeneic with the subject, thereby treating the subject.
• GVHD non-graft versus host
• Tcm central memory T- lymphocyte
• treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
• the term "subject" or “subject in need thereof refers to a mammal, preferably a human being, male or female at any age that is in need of a cell or tissue graft.
• the subject is in need of cell or tissue graft (also referred to herein as recipient) due to a disorder or a pathological or undesired condition, state, or syndrome, or a physical, morphological or physiological abnormality which is amenable to treatment via transplantation of a cell or tissue graft. Examples of such disorders are provided further below.
• tissue or tissue graft refers to a bodily cell (e.g. a single cell or a group of cells) or tissue (e.g. solid tissues or soft tissues, which may be transplanted in full or in part).
• tissue e.g. solid tissues or soft tissues, which may be transplanted in full or in part.
• Exemplary tissues which may be transplanted according to the present teachings include, but are not limited to, lymphoid/hematopoietic tissues (e.g. lymph node, Peyer's patches thymus or bone marrow).
• Exemplary cells which may be transplanted according to the present teachings include, but are not limited to, hematopoietic stem cells (e.g. immature hematopoietic cells).
• the hematopoietic stem cells of the present invention are CD34+.
• the method may be effected using a cell or tissue graft which is syngeneic or non-syngeneic with the subject.
• syngeneic refers to a cell or tissue which is derived from an individual who is essentially genetically identical with the subject. Typically, essentially fully inbred mammals, mammalian clones, or homozygotic twin mammals are syngeneic.
• syngeneic cells or tissues include cells or tissues derived from the subject (also referred to in the art as "autologous"), a clone of the subject, or a homozygotic twin of the subject.
• non-syngeneic refers to a cell or tissue which is derived from an individual who is allogeneic or xenogeneic with the subject's lymphocytes (also referred to in the art as “non-autologous”).
• allogeneic refers to a cell or tissue which is derived from a donor who is of the same species as the subject, but which is substantially non- clonal with the subject. Typically, outbred, non-zygotic twin mammals of the same species are allogeneic with each other. It will be appreciated that an allogeneic donor may be HLA identical or HLA non-identical with respect to the subject.
• xenogeneic refers to a cell or tissue which substantially expresses antigens of a different species relative to the species of a substantial proportion of the lymphocytes of the subject. Typically, outbred mammals of different species are xenogeneic with each other.
• xenogeneic cells or tissues are derived from a variety of species such as, but not limited to, bovines (e.g., cow), equids (e.g., horse), porcines (e.g. pig), ovids (e.g., goat, sheep), felines (e.g., Felis domestica), canines (e.g., Canis domestica), rodents (e.g., mouse, rat, rabbit, guinea pig, gerbil, hamster) or primates (e.g., chimpanzee, rhesus monkey, macaque monkey, marmoset).
• bovines e.g., cow
• equids e.g., horse
• porcines e.g. pig
• ovids e.g., goat, sheep
• felines e.g., Felis domestica
• canines e.g., Canis domestica
• rodents e.g.,
• Cells or tissues of xenogeneic origin are preferably obtained from a source which is known to be free of zoonoses, such as porcine endogenous retroviruses.
• human-derived cells or tissues are preferably obtained from substantially pathogen-free sources.
• both the subject and the donor are humans.
• the cells or tissue grafts of the present invention may be obtained from a prenatal organism, postnatal organism, an adult or a cadaver donor. Moreover, depending on the application needed, the cells or tissues may be naive or genetically modified. Such determinations are well within the ability of one of ordinary skill in the art
• Any method known in the art may be employed to obtain a cell or tissue (e.g. for transplantation).
• Transplanting the cell or tissue graft into the subject may be effected in numerous ways, depending on various parameters, such as, for example, the cell or tissue type; the type, stage or severity of the recipient's disease (e.g. organ failure); the physical or physiological parameters specific to the subject; and/or the desired therapeutic outcome.
• various parameters such as, for example, the cell or tissue type; the type, stage or severity of the recipient's disease (e.g. organ failure); the physical or physiological parameters specific to the subject; and/or the desired therapeutic outcome.
• Transplanting a cell or tissue graft of the present invention may be effected by transplanting the cell or tissue graft into any one of various anatomical locations, depending on the application.
• the cell or tissue graft may be transplanted into a homotopic anatomical location (a normal anatomical location for the transplant), or into an ectopic anatomical location (an abnormal anatomical location for the transplant).
• the cell or tissue graft may be advantageously implanted under the renal capsule, or into the kidney, the testicular fat, the sub cutis, the omentum, the portal vein, the liver, the spleen, the bones, the heart cavity, the heart, the chest cavity, the lung, the skin, the pancreas and/or the intra abdominal space.
• immature autologous, allogeneic or xenogeneic hematopoietic cells e.g. stem cells
• immature autologous, allogeneic or xenogeneic hematopoietic cells which can be derived, for example, from bone marrow, mobilized peripheral blood (by for example leukapheresis), fetal liver, yolk sac and/or cord blood of the syngeneic or non-syngeneic donor can be transplanted to a recipient suffering from a disease.
• the disease is a malignant disease.
• the malignant disease is a malignancy of hematopoietic or lymphoid tissues.
• the malignant disease is a B cell malignancy (i.e. involving B lymphocytes).
• Such a disease includes, but is not limited to, leukemia [e.g., acute lymphatic, acute lymphoblastic, acute lymphoblastic pre-B cell, acute lymphoblastic T cell leukemia, acute - megakaryoblastic, monocytic, acute myelogenous, acute myeloid, acute myeloid with eosinophilia, B cell, basophilic, chronic myeloid, chronic, B cell, eosinophilic, Friend, granulocytic or myelocytic, hairy cell, lymphocytic, megakaryoblastic, monocytic, monocytic-macrophage, myeloblastic, myeloid, myelomonocytic, plasma cell, pre-B cell, promyelocytic, subacute, T cell, lymphoid neoplasm, predisposition to myeloid malignancy, acute nonlymphocytic leukemia, T-cell acute lymphocytic leukemia
• graft rejection chronic graft rejection, subacute graft rejection, hyperacute graft rejection, acute graft rejection and graft versus host disease
• autoimmune diseases such as Type 1 diabetes, severe combined immunodeficiency syndromes (SCID), including adenosine deaminase (ADA), osteopetrosis, aplastic anemia, Gaucher's disease, thalassemia and other congenital or genetically-determined hematopoietic abnormalities.
• SCID severe combined immunodeficiency syndromes
• ADA adenosine deaminase
• osteopetrosis aplastic anemia
• Gaucher's disease thalassemia
• other congenital or genetically-determined hematopoietic abnormalities congenital or genetically-determined hematopoietic abnormalities.
• syngeneic or non-syngeneic hematopoietic cells may be transplanted into a recipient using any method known in the art for cell transplantation, such as but not limited to, cell infusion (e.g. I.V.) or via an intraperitoneal route.
• a cell or tissue graft of the present invention when transplanting a cell or tissue graft of the present invention into a subject having a defective organ, it may be advantageous to first at least partially remove the failed organ from the subject so as to enable optimal development of the graft, and structural/functional integration thereof with the anatomy/physiology of the subject.
• transplantation of the cell or tissue graft into the subject according to the present teachings it is advisable, according to standard medical practice, to monitor the growth functionality and immuno-compatability of the organ according to any one of various standard art techniques.
• structural development of the cells or tissues may be monitored via computerized tomography or ultrasound imaging while engraftment of non-syngeneic cell or bone marrow grafts can be monitored for example by chimerism testing [e.g. by PCR-based procedures using short tandem repeat (STR) analysis].
• STR short tandem repeat
• the method of the present invention utilizes anti-third party Tcm cells.
• the subject is administered a therapeutically effective amount of an isolated population of cells comprising non- graft versus host (GVHD) inducing anti-third party cells having a central memory T- lymphocyte (Tcm) phenotype, the cells being tolerance-inducing cells and capable of homing to the lymph nodes following transplantation.
• GVHD non-graft versus host
• Tcm central memory T- lymphocyte
• isolated population of cells refers to cells which have been isolated from their natural environment (e.g., the human body).
• non-GVHD refers to having substantially no graft versus host inducing reactivity.
• the cells of the present invention are generated as to not significantly cause graft versus host disease (GVHD).
• anti-third party cells refers to lymphocytes (e.g. T lymphocytes) which are directed (e.g. by T cell recognition) against a third party antigen or antigens.
• third party antigen or antigens refers to a soluble or non-soluble (such as membrane associated) antigen or antigens which are not present in either the donor or recipient, as depicted in detail infra.
• the third party antigen or antigens of the present invention is selected from the group consisting of third party cells, a cell antigen, a viral antigen, a bacterial antigen, a protein extract, a purified protein and a synthetic peptide presented by autologous presenting cells, non-autologous presenting cells or on an artificial vehicle or artificial antigen presenting cell.
• third party antigens can be third party cells, antigens of viruses, such as for example, Epstein-Barr virus (EBV) or cyto-megalo virus (CMV) or antigens of bacteria, such as flagellin.
• Viral or bacterial antigens can be presented by cells (e.g., cell line) infected therewith or otherwise made to express viral/bacterial proteins.
• Autologous or non-autologous antigen presenting cells can be used to present short synthetic peptides fused or loaded thereto. Such short peptides may be viral derived peptides or peptides representing any other antigen.
• Dedicated software can be used to analyze viral or other sequences to identify immunogenic short peptides, i.e., peptides presentable in context of class I MHC or class II MHC.
• Third party cells can be either allogeneic or xenogeneic with respects to the recipient (explained in further detail below).
• allogeneic third party cells such cells have HLA antigens different from that of the donor but which are not cross reactive with the recipient HLA antigens, such that anti-third party cells generated against such cells are not reactive against a transplant or recipient antigens.
• the allogeneic or xenogeneic third party cells are stimulatory cells such as, but not limited to, cells purified from peripheral blood lymphocytes (PBL), spleen or lymph nodes, cytokine- mobilized PBLs, in vitro expanded antigen-presenting dendritic cells (APC), B cell lines, Antigen presenting cells (APC) such as artificial APC (e.g. K562 cell line transfected with HLA and/or costimulatory molecules).
• PBL peripheral blood lymphocytes
• APC in vitro expanded antigen-presenting dendritic cells
• B cell lines B cell lines
• Antigen presenting cells such as artificial APC (e.g. K562 cell line transfected with HLA and/or costimulatory molecules).
• the third party antigen or antigens comprise dendritic cells.
• Third party antigens can be presented on the cellular, viral or bacterial surfaces or derived and/or purified therefrom. Additionally, a viral , bacterial or any foreign antigen can be displayed on an infected cell or can be displayed on an artificial vehicle such as a liposome or an artificial APC (e.g.fibroblast or leukemic cell line transfected with the third party antigen or antigens).
• a viral , bacterial or any foreign antigen can be displayed on an infected cell or can be displayed on an artificial vehicle such as a liposome or an artificial APC (e.g.fibroblast or leukemic cell line transfected with the third party antigen or antigens).
• third party antigens can, for example, be proteins extracted or purified from a variety of sources.
• An example of a purified protein which can serve as a third party antigen according to the present invention is ovalbumin.
• graft versus disease e.g. cancer cell such as graft versus leukemia
• LFA1- ICAM1 binding e.g. LFA1- ICAM1 binding
• the anti-third party cells of the present invention comprise a central memory T-lymphocyte (Tcm) phenotype.
• Tcm central memory T-lymphocyte
• central memory T-lymphocyte (Tcm) phenotype refers to a subset of T cytotoxic cells which home to the lymph nodes.
• Cells having the Tcm phenotype in humans, typically express CD8+/CD62L+/CD45R0+/L- selectin+/CD45RA-.
• the Tcm phenotype comprises a CD8+/CD62L+ signature. It will be appreciated that Tcm cells may express all of the signature markers on a single cell or may express only part of the signature markers on a single cell.
• At least at least 30 %, at least 40 %, at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 , at least 90 %, at least 95 % or at least 100 % of the isolated population of cells comprise cells having the Tcm cell signature.
• the Tcm cells typically home to the lymph nodes following transplantation.
• the anti-third party Tcm cells of the present invention may home to any of the lymph nodes following transplantation, as for example, the peripheral lymph nodes and mesenteric lymph nodes.
• the homing nature of these cells allows them to exert their tolerance effect in a rapid and efficient manner.
• the anti-third party Tcm cells of the present invention are tolerance - inducing cells.
• tolerance inducing cells refers to cells which provoke decreased responsiveness of the recipient's cells (e.g. recipient's T cells) when they come in contact with same.
• Tolerance inducing cells include veto cells (i.e. T cells which lead to apoptosis of host T cells upon contact with same) as was previously described in PCT Publication Nos. WO 2001/049243 and WO 2002/102971.
• tolerance inducing cells is especially beneficial in situations in which there is a need to eliminate graft rejection and overcome graft versus host disease (GVHD), such as in transplantation of allogeneic or xenogeneic cells or tissues.
• GVHD graft versus host disease
• the Tcm cells of the present invention may be naive cells (e.g. non-genetically modified) or genetically modified cells (e.g. cells which have been genetically engineered to express or not express specific genes, markers or peptides or to secrete or not secrete specific cytokines). Any method known in the art may be implemented in genetically engineering the cells, such as by inactivation of the relevant gene/s or by insertion of an antisense RNA interfering with polypeptide expression (see e.g. WO/2000/039294, which is hereby incorporated by reference).
• Any method used for the generation of anti-third party non-alloreactive cells (devoid of graft versus host (GVH) activity) can be used in accordance with the present teachings.
• the anti-third party Tcm cells of the present invention are typically generated by first contacting syngeneic or non-syngeneic peripheral blood mononuclear cells (PBMC) with a third party antigen or antigens (such as described above) in a culture deprived of cytokines (i.e.,. without the addition of cytokines). This step is typically carried out for about 12-72 hours, 24-48 hours, 1-10 days, 1-7 days, 1-5 days, 2-3 days or 2 days and allows elimination of GVH reactive cells (e.g. T cells).
• PBMC peripheral blood mononuclear cells
• the anti-third party Tcm cells may be generated devoid of graft versus host (GVH) activity by supplementing the otherwise cytokine-free culture with IL-21 (0.001-3000 ng/ml, 10 - 1000 ng/ml, 10-100 ng/ml, 1-100 ng/ml, 0.1-100 ng/ml, 0.1-10 ng/ml, 1-50 ng/ml or 1- 10 ng/ml). This step is typically carried out for about 12-72 hours, 24-48 hours, 1-10 days, 1-10 days, 1-7 days, 1-5 days, 2-3 days or 3 days.
• IL-21 0.001-3000 ng/ml, 10 - 1000 ng/ml, 10-100 ng/ml, 1-100 ng/ml, 0.1-100 ng/ml, 0.1-10 ng/ml, 1-50 ng/ml or 1- 10 ng/ml.
• the anti-third party cells are cultured in the presence of IL-15 (0.05-500 ng ml, 0.001-3000 ng/ml, 10 - 1000 ng/ml, 10-100 ng/ml, 1-100 ng/ml, 0.1-100 ng/ml, 0.1-10 ng/ml, 1-50 ng/ml or 1-10 ng/ml.
• the concentration is 5 ng/ml) for a period of about 3-30 days, 6-30 days, 3-20 days, 10-20 days, 3-15 days, 5-15 days, 7-15 days, 7-14 days, 3-10 days, 3-7 days or 14 days in an antigen-free environment.
• the culture may be further effected in the presence of additional cytokines such as IL-7 (0.05-500 ng/ml, 0.001-3000 ng/ml, 10 - 1000 ng/ml, 10-100 ng/ml, 1-100 ng/ml, 0.1-100 ng/ml, 0.1-10 ng/ml, 1-50 ng/ml or 1-10 ng/ml.
• additional cytokines such as IL-7 (0.05-500 ng/ml, 0.001-3000 ng/ml, 10 - 1000 ng/ml, 10-100 ng/ml, 1-100 ng/ml, 0.1-100 ng/ml, 0.1-10 ng/ml, 1-50 ng/ml or 1-10 ng/ml.
• the concentration is 5 ng/ml) and/or IL-21 (0.001- 3000 ng/ml, 0.001-3000 ng/ml, 10 - 1000 ng/ml, 10-100 ng/ml, 1-100 ng/ml, 0.1-100 ng/ml, 0.1-10 ng/ml, 1-50 ng/ml or 20-50 ng/ml.
• the concentration is 30 ng/ml). This process enables proliferation of anti-third party cells comprising a central memory T-lymphocyte (Tcm) phenotype and being deprived of GVHD reactivity.
• Tcm central memory T-lymphocyte
• an additional step which allows selection of CD8 + T cells may be carried out, such as by the use of MACS beads, before culturing the cells in the presence of IL-15.
• Such a step may be beneficial in order to increase the purity of the CD8 + cells within the culture (i.e. eliminate other lymphocytes within the cell culture e.g. T CD4 + cells) or in order to increase the number of CD8 + T cells.
• isolation of CD8+ cells can be done prior to culturing with the third party antigen or antigens or following culturing with the third party antigen or antigens and prior to culturing with CD15.
• syngeneic PBMCs e.g. from the subject
• non-syngeneic PBMCs e.g. allogeneic or xenogeneic with respect to the subject
• the source of the PBMCs will be determined with respect to the intended use of the cells (see further details hereinbelow) and is well within the capability of one skilled in the art, especially in light of the detailed disclosure provided herein.
• the anti-third party Tcm cells may be of the same origin as the cell or tissue graft (e.g. bone marrow cells), specifically, they may both be derived from a syngeneic donor (e.g. from the subject, see Example 2 and Figures 1A-I) or may both be derived from a non-syngeneic donor (e.g. from an allogeneic donor, see Example 4 and Figures 3A-I).
• the anti-third party Tcm cells may be from a different origin compared to the cell or tissue graft (e.g. the bone marrow cells may be from the subject and the anti-third party cells may be from an allogeneic donor, Example 3 and Figures 2A-G).
• the anti-third party Tcm cells may be non-syngeneic (e.g. allogeneic or xenogeneic) with both the subject and the graft.
• non-syngeneic e.g. allogeneic or xenogeneic
• the phrase "non-syngeneic with both the subject and the graft" when relating to anti-third party Tcm cells of the present invention qualifies the anti- third party Tcm cells as being allogeneic or xenogeneic with the subject, and allogeneic or xenogeneic with the graft in any combination.
• the anti-third party Tcm cells may be obtained from an origin different from the subject and from the graft donor.
• the anti-third party Tcm cells are non- syngeneic with both the subject and the graft (e.g. from a second donor).
• the anti-third party Tcm cells may be non- syngeneic with respect to only the subject. According to another embodiment, the anti- third party Tcm cells may be non-syngeneic with respect to only the cell or tissue graft.
• the anti-third party Tcm cells are non-syngeneic with the graft and syngeneic with the subject (e.g. of an autologous origin in situations in which the graft is of a non-autologous origin).
• the graft comprises immature hematopoietic cells which are non-syngeneic with the subject (e.g. non-autologous)
• the isolated population of cells are syngeneic with the subject (e.g. autologous).
• Immature hematopoietic cells refers to any type of incompletely differentiated cells which are capable of differentiating into one or more types of fully differentiated hematopoietic cells. Immature hematopoietic cells include without limitation types of cells referred to in the art as “progenitor cells”, “precursor cells”, “stem cells”, “pluripotent cells”, “multipotent cells”, and the like.
• the immature hematopoietic cells are hematopoietic stem cells.
• the immature hematopoietic cells are derived from a human
• the immature hematopoietic cells are CD34+ cells, such as CD34+CD133+ cells.
• Types of grafts of the present invention which comprise immature hematopoietic cells include whole bone marrow cell grafts (T-cell depleted or non-T-cell-depleted), grafts of immature hematopoietic cells from bone marrow aspirates, grafts of peripheral blood-derived immature hematopoietic cells and grafts of umbilical cord-derived immature hematopoietic cells. Methods of obtaining such grafts are described hereinbelow.
• a graft which comprises human peripheral blood-derived hematopoietic stem cells may be obtained according to standard methods, for example by mobilizing CD34+ cells into the peripheral blood by cytokine treatment of the donor, and harvesting of the mobilized CD34+ cells via leukapheresis.
• Ample guidance is provided in the literature of the art for practicing isolation of bone marrow-derived stem cells from the bone marrow or the blood (refer, for example, to: Arai S, Klingemann HG., 2003. Arch Med Res. 34:545-53; and Repka T. and Weisdorf D., 1998. Curr Opin Oncol. 10:112-7; Janssen WE. et al, 1994. Cancer Control 1:225-230; Atkinson K., 1999. Curr Top Pathol. 92:107-36).
• a graft of human umbilical cord blood-derived hematopoietic stem cells may be obtained according to standard methods (refer, for example, to: Quillen K, Berkman EM., 1996. J Hematother. 5:153-5).
• a graft of hematopoietic stem cells of the present invention may also be derived from liver tissue or yolk sac.
• a requisite number of hematopoietic ste cells can be provided by ex- vivo expansion of primary hematopoietic stem cells (reviewed in Emerson, 1996, Blood 87:3082, and described in more detail in Petzer et al., 1996, Proc. Natl. Acad. Sci. U. S. A. 3:1470; Zundstra et al., 1994, BioTechnology 12:909; and WO 95 11692).
• the graft comprises immature hematopoietic cells which are non-syngeneic with the subject (e.g. non-autologous)
• the isolated population of cells are non-syngeneic with both the subject and with the graft. (e.g. the immature hematopoietic cells and the isolated population of cells are from different donors).
• a method of treating a disease in a subject in need thereof comprising: (a) transplanting immature hematopoietic cells to the subject; and (b) administering to the subject a therapeutically effective amount of an isolated population of cells comprising non-graft versus host (GVHD) inducing anti-third party cells having a central memory T-lymphocyte (Tcm) phenotype, the cells being tolerance-inducing cells and capable of homing to the lymph nodes following transplantation, wherein when the immature hematopoietic cells are syngeneic with the subject, the isolated population of cells are selected syngeneic with the subject or non-syngeneic with the subject.
• GVHD non-graft versus host
• Tcm central memory T-lymphocyte
• both the immature hematopoietic cells and the isolated population of cells are autologous (e.g. from the subject).
• the immature hematopoietic cells are autologous (e.g. from the subject) and the isolated population of cells are non-autologous (e.g. from a donor).
• a method of treating a disease in a subject in need thereof comprising: (a) transplanting immature hematopoietic cells to the subject; and (b) administering to the subject a therapeutically effective amount of an isolated population of cells comprising non-graft versus host (GVHD) inducing anti-third party cells having a central memory T-lymphocyte (Tcm) phenotype, the cells being tolerance-inducing cells and capable of homing to the lymph nodes following transplantation, wherein when the immature hematopoietic cells are non-syngeneic with the subject, the isolated population of cells are selected syngeneic with the subject or non-syngeneic with both the subject and the immature hematopoietic cells.
• GVHD non-graft versus host
• Tcm central memory T-lymphocyte
• the immature hematopoietic cells are non- autologous (e.g. from a donor) and the isolated population of cells are autologous (e.g. from the subject).
• the anti-third party Tcm cells are non-syngeneic with both the subject and the graft (e.g. two different donors).
• the immature hematopoietic cells and the isolated population of cells are from different donors.
• an isolated population of cells comprising non-graft versus host GVHD) inducing anti- third party cells having a central memory T-lymphocyte (Tcm) phenotype, the cells being tolerance-inducing cells and capable of homing to the lymph nodes following transplantation, and further wherein the cells are non-syngeneic with both a subject and a cell or tissue graft. (i) non-syngeneic with both a host subject and a graft; or
• the cells non-syngeneic with the graft and syngeneic with the host subject are autologous.
• the present invention contemplates administration to a subject any anti- third party Tcm cells (e.g. non-syngeneic with both the subject and the graft or non- syngeneic with the graft and syngeneic with the subject) which will result in eradication of a disease (e.g. leukemia or lymphoma) and will concomitantly enhance engraftment of a cell or tissue transplant (e.g. autologous or non-autologous bone marrow cells) by being tolerogenic cells and non-GVHD.
• a disease e.g. leukemia or lymphoma
• a cell or tissue transplant e.g. autologous or non-autologous bone marrow cells
• the anti-third party cells may be administered concomitantly with a cell or tissue graft (e.g. as an adjuvant therapy), may be administered prior to transplantation of a cell or tissue graft (e.g. in order to eradicate residual cancer cells prior to transplantation and to eliminate graft rejection and graft versus host disease), or may be administered following transplantation of a cell or tissue graft (e.g. in order to eradicate residual cancer cells following transplantation and to eliminate graft rejection and graft versus host disease).
• a cell or tissue graft e.g. as an adjuvant therapy
• the anti-third party cells may be administered at any time following transplantation.
• the anti-third party Tcm cells are administered on day 0, day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8 or day 10 following transplantation.
• the anti-third party Tcm cells may be administered extended times after transplantation, as for example, two weeks, a month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months or 24 months following transplantation.
• the anti third party Tcm cells may be administered via any method known in the art for cell transplantation, such as but not limited to, cell infusion (e.g. I.V.) or via an intraperitoneal route.
• cell infusion e.g. I.V.
• intraperitoneal route e.g. I.V.
• a therapeutically effective amount is an amount of anti-third party Tcm cells efficient for tolerization, anti-tumor effect and/or immune reconstitution without inducing GVHD. Since the Tcm cells of the present invention home to the lymph nodes following transplantation, lower amounts of cells (compared to the dose of cells previously used, see for example WO 2001/049243) may be needed to achieve the beneficial effect/s of the cells (e.g. tolerization, anti-tumor effect and/or immune reconstitution). It will be appreciated that lower levels of immunosuppressive drugs may be needed in conjunction with the Tcm cells of the present invention (such as exclusion of rapamycin from the therapeutic protocol).
• the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
• a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
• the number of anti-third party Tcm cells infused to a recipient should be more than 1 x 10 4 /Kg body weight.
• the number of anti- third party Tcm cells infused to a recipient should typically be in the range of 1 x 10 4 /Kg body weight to 1 x 10 9 /Kg body weight.
• the method may further advantageously comprise conditioning the subject with an immunosuppressive regimen prior to, concomitantly with, or following transplantation of the cell or tissue graft.
• the subject is conditioned under sublethal, lethal or supralethal conditions prior to transplantation of a cell or tissue graft.
• the subject may be treated with a myeloablative or non- myeloablative conditioning.
• the type of conditioning may be determined by one of ordinary skill in the art and takes into account the age and disease severity of the subject.
• an elderly subject e.g. one who is over 40 years of age
• immunosuppressive regimens include administration of immunosuppressive drugs, tolerance inducing cell populations (as described in detail hereinabove), and/or immunosuppressive irradiation.
• suitable types of immunosuppressive regimens include administration of immunosuppressive drugs, tolerance inducing cell populations (as described in detail hereinabove), and/or immunosuppressive irradiation.
• Ample guidance for selecting and administering suitable immunosuppressive regimens for transplantation is provided in the literature of the art (for example, refer to: Kirkpatrick CH. and Rowlands DT Jr., 1992. JAMA. 268, 2952; Higgins RM. et al.,
• the immunosuppressive regimen consists of administering at least one immunosuppressant agent to the subject.
• immunosuppressive agents include, but are not limited to, methotrexate, cyclophosphamide, cyclosporine, cyclosporin A, chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine), gold salts, D-penicillamine, leflunomide, azathioprine, anakinra, infliximab (REMICADE), etanercept, TNF.alpha. blockers, a biological agent that targets an inflammatory cytokine, and Non-Steroidal Anti-Inflammatory Drug (NSAIDs).
• methotrexate cyclophosphamide
• cyclosporine cyclosporin A
• chloroquine hydroxychloroquine
• sulfasalazine sulphasalazopyrine
• gold salts gold salts
• D-penicillamine leflunomide
• azathioprine anakin
• NSAIDs include, but are not limited to acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, tramadol, rapamycin (sirolimus) and rapamycin analogs (such as CCI-779, RADOOl, AP23573). These agents may be administered individually or in combination.
• compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
• a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
• range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
• method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
• NUDE mice were obtained from Harlan Laboratories. Progeny of B6-NUDE mice were bred at the Weizmann Institute Animal Center. All mice were kept in small cages (5 animals in each cage) and fed sterile food and acid water. All studies were approved by the Weizmann Institute of Science Institutional Animal Care and Use Committee.
• Anti-third-party Tcm were prepared as previously described [Ophir E et al., Blood (2010) 115: 2095-2104] briefly, splenocytes of the donor mice were cultured against irradiated third-party splenocytes for 60 hours under cytokine deprivation. Subsequently, CD8 + cells were positively selected using Magnetic Particles (BD Pharmingen) and cultured in an Ag-free environment. rhIL-15 (20 ng/mL; R&D Systems) was added every second day. To attain a purified population at the end of the culture (day 16), the Tcm cells were positively selected for CD62L expression by magnetic-activated cell sorting [MACS, Milteny, Bergisch Gladbach, Germany]. Flow cytometric analysis
• Fluorescence-activated cell sorting (FACS) analysis was performed using a modified Becton Dickinson FACScan. Cells were stained with labeled antibodies specific for CD8-phycoerythrin (PE)/fluorescein isothiocyanate (FITQ/allophycocyanin (APC) (BD Pharmingen), CalceinAM (Molecular Probes, INC., Eugene, OR, USA). Annexin V and 7-amino-actinomycin D (7AAD) staining were done according to the manufacturer's instructions (BD Pharmingen).
• Anti-3 rd -party Tcm and lymphoma cells were obtained by Ficoll density gradient centrifugation and lymphoma cells were labeled with 0.15 g/ml CalceinAM (Molecular Probes, INC., Eugene, OR, USA) according to manufacturer's instructions and brought to a concentration of 1 x 10 6 cells/ml in the required media. 3 x 10 5 of the lymphoma cells were incubated with anti-3 rd -party CTLs according to the indicated ratios and time intervals for 24-well plates. Cells were recovered and analyzed for survival using surface markers such as Annexin-V (BD) and by measuring the number of CalceinAM stained lymphoma cells by FACS.
• BD Annexin-V
• Annexin V APC was used to detect apoptotic cells. Samples from in-vitro cultures were incubated with a mixture of selected monoclonal antibodies labeled with different fluorochromes for 20 minutes at 4 °C. After washing off the unbound free antibody using an Annexin-V binding buffer, samples were supplemented with 5 ⁇ Annexin V-APC (BD). Cells were then incubated at room temperature for 15 minutes in the dark and washed with an Annexin-V binding buffer. Samples were analyzed by FACS for live cells that are positively stained of AnnexinV.
• BD Annexin V-APC
• mice On the subsequent day (day +1) mice received or did not receive 10 x 10 6 BALB/c derived anti-3 rd -party Tcm (syngeneic) or 5 x 10 6 C57BL/6 derived anti-3 rd -party Tcm (allogeneic), intravenously. Tumor localization, migratory patterns of A20 cells and the anti-lymphoma reactivity of anti-3 rd -party CTLs was surveyed using an in-vivo imaging system.
• mice were anaesthized with Ketamine (100 mg/kg intra- peritoneally (i.p) (Kepro Holand Netherlands) and Xylazine (Kepro Holand Netherlands) (20 mg/Kg i.p), and an aqueous solution of D Luciferin (150 mg/Kg i.p) (Cat#XR-1001, 30 mg/ml in PBS; Xenogen) was injected 10 minutes prior to imaging. Animals were placed into the light-tight chamber of the In Vivo Imaging system (IVIS® 100, Xenogen) coupled with a Pixelfly QE (PCO, K, Germany) charge-coupled device (CCD) camera at the Department of Veterinary Resources of the Weizmann Institute. A grayscale body surface reference image (digital photograph) was taken after a 10 second exposure, under strong illumination. Image data processing and analysis were performed using Living Image 2.5 software. The mice were monitored for tumor growth from day 14 on weekly intervals.
• luciferase expressing A20 cells inventors were able to follow the fate of the malignant cells in vivo, and study the anti-lymphoma effect of added donor anti-3 rd party Tcm to either syngeneic or allogeneic bone marrow transplant (BMT, hereinbelow), in a model simulating minimal residual disease.
• BMT bone marrow transplant
• 3 x 10 6 Nude BALB/c BM cells were transplanted into lethally irradiated BALB/c mice together with 5000 A20 cells. On the next day, syngeneic Tcm were infused.
• mice Similar to the results in the syngeneic model (described in Examples 2 and 3, above), none of the untreated mice survived 100 days post BMT (0/8, 23 days median survival, Figures 3A-D and 31), while administration of 5 x 10 6 donor type Tcm cells, led to remarkable overall survival of 100 % (7/7) 100 days post BMT ( Figures 3E-I). Although the allogeneic Tcm cells displayed enhanced GVL activity compared to syngeneic Tcm cells, this effect was not associated with any manifestation of GVHD. Thus, as previously described, the weight and overall appearance of mice receiving allogeneic anti-3 rd party Tcm cells were the same as that of mice in the control group, radio-protected with a transplant of Nude BM alone.
• PBMCs Peripheral blood mononuclear cells
• Donor's Frozen PBMCs were thawed quickly in a 37 °C water bath and transferred to warm thawing medium (Cellgro DC supplemented with 10 % human serum and Pen/Strep medium supplemented with Benzonase® Nuclease to avoid cell clump formation as a result of dying cells). Thawed donor's PBMCs were washed twice with warm thawing medium.
• PBMCs were then resuspended in culture medium (Cellgro DC supplemented with 5 % human Serum and Pen/Strep and 10 ng/ml IL-7) and were plated on specially coated 6-well plates for overnight incubation at 37 °C.
• culture medium Cellgro DC supplemented with 5 % human Serum and Pen/Strep and 10 ng/ml IL-7
• CD8+ T-cells were isolated using the CD8 isolation kit from Miltenyi in accordance with the manufacturer's protocol. Thereafter, CD8 T cells with naive phenotype were obtained from the total CD8 population by depleting cells expressing the activation marker CD45RO using CD45RO magnetic beads.
• DCs Monocyte derived dendritic cells
• Monocyte derived DCs were generated from allogeneic cryopreserved PBMC.
• the monocytes were enriched from the PBMC by plastic adherence and cultured in GM-CSF and IL-4 over the course of 3 days. Maturation was induced over the final 24 hours of culture with the addition of LPS and IFN- ⁇ .
• Generation of anti-third party Tcm cells (naive CD8 T cells targeting monocyte derived DCs)
• Naive CD8+ T cells were activated with irradiated (30 gy) monocyte derived DCs at a ratio of 1:0.25, in culture medium (Cellgro DC supplemented with 5 % humaSerum and Pen/Strep) supplemented with IL-21 for 3 days. Thereafter, the CD8+ T cells received no further activation and were grown with IL-7 and IL-15 until day 12.
• cell composition and cell phenotype was evaluated using FACS analysis and cell number was determined by trypan blue exclusion.
• the CD8+ T cells comprised largely of 76 % Tcm cells, 9 % Na ' ive cells, 8.5 % Teff/Tem and 6.5 % Temra.
• B cell lines were labeled with 0.15 g/ml CalceinAM, a vital dye that is released upon cell death, according to manufacturer's instructions.
• CalceinAM a vital dye that is released upon cell death
• 0.3 xlO 6 Calcein labeled cell lines were incubated with or without 1.5 xlO 6 anti-3 rd party Tcm for 22 hours in 24 well plates. No exogenous cytokines were added to the MLR. After 22 hours Cells were recovered and analyzed for survival by measuring the number of surviving Calcein stained cells by FACS.
• AnnexinV staining samples were incubated with 5 ⁇ AnnexinV -APC (BD) for 10 minutes at room temperature. Subsequently, unbound AnnexinV was washed out, and samples were analyzed by FACS.
• BD AnnexinV -APC

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